Bacterial extracellular vesicles

ABSTRACT

Provided herein are methods and compositions related to EVs useful as therapeutic agents.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Applications having Ser. No. 62/556,015, filed Sep. 8, 2017, and62/669,151, filed May 9, 2018, the contents of each of which are herebyincorporated herein by reference in their entirety.

SUMMARY

In certain aspects, provided herein are pharmaceutical compositionscomprising bacterial extracellular vesicles (EVs) useful for thetreatment and/or prevention of disease (e.g., cancer, autoimmunedisease, inflammatory disease, metabolic disease), as well as methods ofmaking and/or identifying such EVs, and methods of using suchpharmaceutical compositions (e.g., for the treatment of cancers,autoimmune diseases, inflammatory diseases, metabolic diseases, eitheralone or in combination with other therapeutics). In some embodiments,the pharmaceutical compositions comprise both EVs and whole bacteria(e.g., live bacteria, killed bacteria, attenuated bacteria). In certainembodiments, provided herein are pharmaceutical compositions comprisingbacteria in the absence of EVs. In some embodiments, the pharmaceuticalcompositions comprise EVs in the absence of bacteria. In someembodiments, the pharmaceutical compositions comprise EVs and/orbacteria from one or more of the bacteria strains or species listed inTable 1 and/or Table 2.

In certain embodiments, the pharmaceutical composition comprises aspecific ratio of bacteria to EV particles. For example, in someembodiments, the pharmaceutical composition comprises at least 1bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10¹² EV particles. In some embodiments, the pharmaceutical compositioncomprises about 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³,2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴,3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵,4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10 ¹⁰, 3×10¹⁰, 4×10¹⁰, 5 ×10¹⁰, 6×10¹⁰ , 7×10¹⁰,8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹,8×10 ¹¹, 9×10¹¹, and/or 1×10¹² EV particles. In some embodiments, thepharmaceutical composition comprises no more than 1 bacterium for every1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4,8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³,8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴,9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵,1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷,2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸,3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹,4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹,4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² EVparticles. In some embodiments, the pharmaceutical composition comprisesat least 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, and/or 1×10¹² bacterium. In some embodiments, the pharmaceuticalcomposition comprises about 1 EV particle for every 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴,2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵,3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶,4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷,5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸,6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹,7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰,7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹,7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² bacterium. In some embodiments,the pharmaceutical composition comprises no more than 1 EV particle forevery 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3,8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98.99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³,7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴,8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵,9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶,1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸,2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹,3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹, 2×10¹⁰, 3×10¹⁰,4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×100, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹,4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² bacterium.[4] In certain aspects, the EVs are from an engineered bacteria that ismodified to enhance certain desirable properties. For example, in someembodiments, the engineered bacteria are modified to increase productionof EVs. In some embodiments, the engineered bacteria are modified toproduce EVs with enhanced oral delivery (e.g., by improving acidresistance, muco-adherence and/or penetration and/or resistance to bileacids, resistance to anti-microbial peptides and/or antibodyneutralization), to target desired cell types (e.g. M-cells, gobletcells, enterocytes, dendritic cells, macrophages) to improvebioavailability systemically or in an appropriate niche (e.g.,mesenteric lymph nodes, Peyer's patches, lamina propria, tumor draininglymph nodes, and/or blood), to enhance the immunomodulatory and/ortherapeutic effect of the EVs they produce (e.g., either alone or incombination with another therapeutic agent), to enhance immuneactivation by the EVs they produce and/or to improve bacterial and/or EVmanufacturing (e.g., greater stability, improved freeze-thaw tolerance,shorter generation times). In some embodiments, provided herein aremethods of making such EVs and bacteria.

In certain embodiments, provided herein are methods of treating asubject who has cancer comprising administering to the subject apharmaceutical composition described herein. In certain embodiments,provided herein are methods of treating a subject who has an immunedisorder (e.g., an autoimmune disease, an inflammatory disease, anallergy) comprising administering to the subject a pharmaceuticalcomposition described herein. In certain embodiments, provided hereinare methods of treating a subject who has a metabolic disease comprisingadministering to the subject a pharmaceutical composition describedherein.

In some embodiments, the method further comprises administering to thesubject an antibiotic. In some embodiments, the method further comprisesadministering to the subject one or more other cancer therapies (e.g.,surgical removal of a tumor, the administration of a chemotherapeuticagent, the administration of radiation therapy, and/or theadministration of a cancer immunotherapy, such as an immune checkpointinhibitor, a cancer-specific antibody, a cancer vaccine, a primedantigen presenting cell, a cancer-specific T cell, a cancer-specificchimeric antigen receptor (CAR) T cell, an immune activating protein,and/or an adjuvant). In some embodiments, the method further comprisesthe administration of another therapeutic bacterium and/or EV. In someembodiments, the method further comprises the administration of animmune suppressant and/or an anti-inflammatory agent. In someembodiments, the method further comprises the administration of ametabolic disease therapeutic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the efficacy of iv administered Blautia massiliensiscompared to that of intraperitoneally (i.p.) administered anti-PD-1 in amouse colorectal carcinoma model.

FIG. 2 shows inhibition of tumor growth (by volume) by the ivadministration of Blautia massiliensis compared to intraperitoneally(i.p.) administered anti-PD-1 in a mouse colorectal carcinoma model.

FIG. 3A shows the efficacy of orally or intra-peritoneally administeredPrevotella histicola and P. histicola-derived EVs in reducingantigen-specific ear swelling (ear thickness) 24 hours after antigenchallenge in a KLH-based delayed type hypersensitivity mouse model.Efficacy was seen in both the oral and i.p. administration groups.

FIG. 3B shows the efficacy of orally or intra-peritoneally administeredPrevotella histicola and P. histicola-derived EVs in reducingantigen-specific ear swelling (ear thickness) 48 hours after antigenchallenge in a KLH-based delayed type hypersensitivity mouse model.

FIG. 3C shows the efficacy of intra-peritoneally administered P.histicola-derived EVs at the dose indicated (10 μg, 3 μg, 0.1 μg, and0.1μg) in reducing antigen-specific ear swelling (ear thickness) 48hours after antigen challenge in a KLH-based delayed typehypersensitivity mouse model.

FIG. 3D shows the ability of intra-peritoneally administered Prevotellahisticola-derived EVs to reduce expression of IL-1β 48 hours afterantigen challenge in a KLH-based delayed type hypersensitivity mousemodel.

FIG. 3E shows the ability of intra-peritoneally administered Prevotellahisticola-derived EVs to increase accumulation of Tregs in the cervicallymph nodes 48 hours after antigen challenge in a KLH-based delayed typehypersensitivity mouse model.

FIG. 4 shows that P. histicola was efficacious at reducing the NASHactivity score (NAS) in mice receiving a methionine choline deficient(MCD) diet, which induces NASH symptoms.

FIG. 5A shows that P. histicola reduced steatosis in mice that were fedan MCD diet.

FIG. 5B and FIG. 5C show that P. histicola reduced inflammation in micethat were fed an MCD diet.

FIG. 5D shows that P. histicola reduced ballooning in mice that were fedan MCD diet.

FIG. 6 shows that P. histicola reduced hepatic total cholesterol in micethat were fed an MCD diet.

FIG. 7A and FIG. 7B show that P. histicola reduced the fibrosis score inmice that were fed an MCD diet.

FIG. 8A shows the efficacy of administering Pevotella histicola, P.melanogenica, P. histicola-derived EVs, P. melanogenica-derived EVs inreducing antigen-specific ear swelling (ear thickness) 24 hours afterantigen challenge in a KLH-based delayed type hypersensitivity mousemodel. P. melanogenica-derived EVs is more effective than P.melanogenica.

FIG. 8B shows the efficacy of administering Prevotella histicola, P.melanogenica, P. histicola-derived EVs, P. melanogenica-derived EVs inreducing antigen-specific ear swelling (ear thickness) 48 hours afterantigen challenge in a KLH-based delayed type hypersensitivity mousemodel. P. melanogenica-derived EVs is more effective than P.melanogenica.

FIG. 9A shows the effect of P. histicola on hepatic free fatty acids inmice that were fed an MCD diet.

FIG. 9B shows the effect of P. histicola on hepatic total cholesterol inmice that were fed an MCD diet.

FIG. 9C shows the effect of P. histicola on hepatic triglycerides inmice that were fed an MCD diet.

FIG. 9D shows the effect of P. histicola and P. melanogenicaon alanineaminotransferase in mice that were fed an MCD diet.

FIG. 9E shows the effect of P. histicola and P. melanogenica onaspartate aminotransferase in mice that were fed an MCD diet.

FIG. 10A shows that P. histicola is efficacious at reducing the NASHactivity score (NAS) in mice receiving a methionine choline deficient(MCD) diet, which induces NASH symptoms.

FIG. 10B shows that Prevotella histicola, P. melanogenica, P. histicolain combination with OCD at reducing the NASH activity score (NAS) inmice receiving a methionine choline deficient (MCD) diet, which inducesNASH symptoms.

FIG. 11 shows the efficacy of orally administered Veillonellatobetsuensis and Veillonella parvula Strains in reducingantigen-specific ear swelling (ear thickness) at 24 hours compared tovehicle (negative control), anti-inflammatory Dexamethasone (positivecontrol), and Bifidobacterium animalis lactis in a KLH-based delayedtype hypersensitivity mouse model.

FIG. 12 shows the efficacy of EVs from two Veillonella tobetsuensis andVeillonella parvula strains compared to intraperitoneal injected (i.p.)anti-PD-1 or vehicle in a mouse colorectal carcinoma model.

FIG. 13 shows the efficacy of EVs from Veillonella tobetsuensis andVeillonella parvula Strains compared to intraperitoneal injected (i.p.)anti-PD-1 or vehicle in a mouse colorectal carcinoma model at day 11.

FIG. 14 shows the dose and route of administration dependent efficacy ofEVs from Veillonella tobetsuensis and Veillonella parvula strainscompared to intraperitoneal injected (i.p.) anti-PD-1 or vehicle in amouse colorectal carcinoma model.

FIG. 15 shows the dose and route of administration dependent efficacy ofEVs from Veillonella tobetsuensis and Veillonella parvula strainscompared to intraperitoneal injected (i.p.) anti-PD-1 or vehicle in amouse colorectal carcinoma model at day 11.

FIG. 16 shows that Veillonella parvula was efficacious at reducing theNASH activity score (NAS) in mice receiving a methionine cholinedeficient (MCD) diet, which induces NASH symptoms.

FIG. 17 shows that Veillonella parvula reduced Fibrosis in mice thatwere fed an MCD diet.

FIG. 18 shows that Veillonella parvula reduced Hepatic Total Cholesterolin mice that were fed an MCD diet.

FIG. 19 shows that Veillonella parvula reduced Hepatic Triglycerides inmice that were fed an MCD diet

FIG. 20 shows the efficacy of Burkholderia pseudomallei EVs compared tointravenously (i.v.) administered anti-PD-1 or vehicle in a mousecolorectal carcinoma model.

FIG. 21 shows the efficacy of Burkholderia pseudomallei EVs compared tointravenously (i.v.) administered anti-PD-1 or vehicle in a mousecolorectal carcinoma model at day 11. ** The difference in tumor volumein the Burkholderia pseudomallei EV treated group compared to vehiclecontrol group on day 11 was highly significant, with a P-value of 0.0011as determined by T-test (two-tailed, unpaired, welch-corrected)calculated in GraphPad.

FIG. 22 shows the efficacy of Neisseria Meningitidis EVs compared tothat of intraperitoneally (i.p.) administered anti-PD-1 or vehicle in amouse colorectal carcinoma model.

FIG. 23 shows the efficacy of Neisseria Meningitidis EVs compared tothat of intraperitoneally (i.p.) administered anti-PD-1 or vehicle in amouse colorectal carcinoma model at day 11.

DETAILED DESCRIPTION Definitions

“Adjuvant” or “Adjuvant therapy” broadly refers to an agent that affectsan immunological or physiological response in a patient or subject. Forexample, an adjuvant might increase the presence of an antigen over timeor to an area of interest like a tumor, help absorb an antigenpresenting cell antigen, activate macrophages and lymphocytes andsupport the production of cytokines. By changing an immune response, anadjuvant might permit a smaller dose of an immune interacting agent toincrease the effectiveness or safety of a particular dose of the immuneinteracting agent. For example, an adjuvant might prevent T cellexhaustion and thus increase the effectiveness or safety of a particularimmune interacting agent.

“Administration” broadly refers to a route of administration of acomposition to a subject. Examples of routes of administration includeoral administration, rectal administration, topical administration,inhalation (nasal) or injection. Administration by injection includesintravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous(SC) administration. The pharmaceutical compositions described hereincan be administered in any form by any effective route, including butnot limited to intratumoral, oral, parenteral, enteral, intravenous,intraperitoneal, topical, transdermal (e.g., using any standard patch),intradermal, ophthalmic, (intra)nasally, local, non-oral, such asaerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual,(trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal(e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal(e.g., trans- and perivaginally), implanted, intravesical,intrapulmonary, intraduodenal, intragastrical, and intrabronchial. Inpreferred embodiments, the pharmaceutical compositions described hereinare administered orally, rectally, intratumorally, topically,intravesically, by injection into or adjacent to a draining lymph node,intravenously, by inhalation or aerosol, or subcutaneously.

As used herein, the term “antibody” may refer to both an intact antibodyand an antigen binding fragment thereof. Intact antibodies areglycoproteins that include at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain includesa heavy chain variable region (abbreviated herein as VH) and a heavychain constant region. Each light chain includes a light chain variableregion (abbreviated herein as VL) and a light chain constant region. TheVH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The term “antibody” includes, for example, monoclonalantibodies, polyclonal antibodies, chimeric antibodies, humanizedantibodies, human antibodies, multispecific antibodies (e.g., bispecificantibodies), single-chain antibodies and antigen-binding antibodyfragments.

The terms “antigen binding fragment” and “antigen-binding portion” of anantibody, as used herein, refers to one or more fragments of an antibodythat retain the ability to bind to an antigen. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of anantibody include Fab, Fab′, F(ab′)₂, Fv, scFv, disulfide linked Fv, Fd,diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, andother antibody fragments that retain at least a portion of the variableregion of an intact antibody. These antibody fragments can be obtainedusing conventional recombinant and/or enzymatic techniques and can bescreened for antigen binding in the same manner as intact antibodies.

“Cancer” broadly refers to an uncontrolled, abnormal growth of a host'sown cells leading to invasion of surrounding tissue and potentiallytissue distal to the initial site of abnormal cell growth in the host.Major classes include carcinomas which are cancers of the epithelialtissue (e.g., skin, squamous cells); sarcomas which are cancers of theconnective tissue (e.g., bone, cartilage, fat, muscle, blood vessels,etc.); leukemias which are cancers of blood forming tissue (e.g., bonemarrow tissue); lymphomas and myelomas which are cancers of immunecells; and central nervous system cancers which include cancers frombrain and spinal tissue. “Cancer(s),” “neoplasm(s),” and “tumor(s)” areused herein interchangeably. As used herein, “cancer” refers to alltypes of cancer or neoplasm or malignant tumors including leukemias,carcinomas and sarcomas, whether new or recurring. Specific examples ofcancers are: carcinomas, sarcomas, myelomas, leukemias, lymphomas andmixed type tumors. Non-limiting limiting examples of cancers are new orrecurring cancers of the brain, melanoma, bladder, breast, cervix,colon, head and neck, kidney, lung, non-small cell lung, mesothelioma,ovary, prostate, sarcoma, stomach, uterus and medulloblastoma.

“Cellular augmentation” broadly refers to the influx of cells orexpansion of cells in an environment that are not substantially presentin the environment prior to administration of a composition and notpresent in the composition itself. Cells that augment the environmentinclude immune cells, stromal cells, bacterial and fungal cells.Environments of particular interest are the microenvironments wherecancer cells reside or locate. In some instances, the microenvironmentis a tumor microenvironment or a tumor draining lymph node. In otherinstances, the microenvironment is a pre-cancerous tissue site or thesite of local administration of a composition or a site where thecomposition will accumulate after remote administration.

“Clade” refers to the OTUs or members of a phylogenetic tree that aredownstream of a statistically valid node in a phylogenetic tree. Theclade comprises a set of terminal leaves in the phylogenetic tree thatis a distinct monophyletic evolutionary unit and that share some extentof sequence similarity. “Operational taxonomic units,” “OTU” (or plural,“OTU”) refer to a terminal leaf in a phylogenetic tree and is defined bya nucleic acid sequence, e.g., the entire genome, or a specific geneticsequence, and all sequences that share sequence identity to this nucleicacid sequence at the level of species. In some embodiments the specificgenetic sequence may be the 16S sequence or a portion of the I 6Ssequence. In other embodiments, the entire genomes of two entities aresequenced and compared. In another embodiment, select regions such asmultilocus sequence tags (MLST), specific genes, or sets of genes may begenetically compared. in 16S embodiments, OTUs that share ≥97% averagenucleotide identity across the entire 16S or some variable region of the16S are considered the same OUT (see e.g. Claesson M J, Wang Q,O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010.Comparison of two next-generation sequencing technologies for resolvinghighly complex microbiota composition using tandem variable 16S rRNAgene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A,and Tiedje J M. 2006, The bacterial species definition in the genomicera. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). In embodimentsinvolving the complete genome, MLSTs, specific genes, or sets of genesOTUs that share ≥95% average nucleotide identity are considered the sameOTU (see e.g. Achtman M, and Wagner M. 2008. Microbial diversity and thegenetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440.Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterialspecies definition in the genomic era. Philos Trans R Soc Lond Biol Sci361: 1929-1940.). OTUs are frequently defined by comparing sequencesbetween organisms. Generally, sequences with less than 95% sequenceidentity are not considered to form part of the same OTU OTUs may alsobe characterized by any combination of nucleotide markers or genes, inparticular highly conserved genes (e.g., “house-keeping” genes), or acombination thereof. Such characterization employs, e.g.,WGS data or awhole genome sequence. [49] A “combination” of EVs from two or moremicrobial strains includes the physical co-existence of the two EVs,either in the same material or product or in physically connectedproducts, as well as the temporal co-administration or co-localizationof the EVs from the two strains.

The term “decrease” or “deplete” means a change, such that thedifference is, depending on circumstances, at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000or undetectable after treatment when compared to a pre-treatment state.

As used herein, the term “Dysbiosis” refers to a state in which thesynergy between microbes and the tumor is broken such as the microbes nolonger support the nucleation, maintenance, progression or spread ormetastasis of a tumor.

The term “epitope” means a protein determinant capable of specificbinding to an antibody or T cell receptor. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains. Certain epitopes can be defined by a particularsequence of amino acids to which an antibody is capable of binding.

As used herein, “engineered bacteria” are any bacteria that have beengenetically altered from their natural state by human intervention andthe progeny of any such bacteria. Engineered bacteria include, forexample, the products of targeted genetic modification, the products ofrandom mutagenesis screens and the products of directed evolution.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. The term “gene” applies to a specificgenomic sequence, as well as to a cDNA or an mRNA encoded by thatgenomic sequence.

“Identity” as between nucleic acid sequences of two nucleic acidmolecules can be determined as a percentage of identity using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to HugeComputers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, andCarillo et al. (1988) SIAM J Applied Math 48:1073). For example, theBLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.)).

As used herein, the term “immune disorder” refers to any disease,disorder or disease symptom caused by an activity of the immune system,including autoimmune diseases, inflammatory diseases and allergies.Immune disorders include, but are not limited to, autoimmune diseases(e.g., Lupus, Scleroderma, hemolytic anemia, vasculitis, type onediabetes, Grave's disease, rheumatoid arthritis, multiple sclerosis,Goodpasture's syndrome, pernicious anemia and/or myopathy), inflammatorydiseases (e.g., acne vulgaris, asthma, celiac disease, chronicprostatitis, glomerulonephritis, inflammatory bowel disease, pelvicinflammatory disease, reperfusion injury, rheumatoid arthritis,sarcoidosis, transplant rejection, vasculitis and/or interstitialcystitis), and/or an allergies (e.g., food allergies, drug allergiesand/or environmental allergies).

“Immunotherapy” is treatment that uses a subject's immune system totreat disease (e.g., immune disease, inflammatory disease, metabolicdisease, cancer) and includes, for example, checkpoint inhibitors,cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendriticcell therapy.

The term “increase” means a change, such that the difference is,depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10{circumflex over ( )}3fold, 10{circumflex over ( )}4 fold, 10{circumflex over ( )}5 fold,10{circumflex over ( )}6 fold, and/or 10{circumflex over ( )}7 foldgreater after treatment when compared to a pre-treatment state.Properties that may be increased include immune cells, bacterial cells,stromal cells, myeloid derived suppressor cells, fibroblasts,metabolites, and cytokines.

“Innate immune agonists” or “immuno-adjuvants” are small molecules,proteins, or other agents that specifically target innate immunereceptors including Toll-Like Receptors (TLR), NOD receptors, RLRs,C-type lectin receptors, STING-cGAS Pathway components, inflammasomecomplexes. For example, LPS is a TLR-4 agonist that is bacteriallyderived or synthesized and aluminum can be used as an immune stimulatingadjuvant. immuno-adjuvants are a specific class of broader adjuvant oradjuvant therapy. Examples of STING agonists include, but are notlimited to, 2′3′- cGAMP, 3′3′-cGAMP, c-di-AMP, 2′2′-cGAMP, and2′3′-cGAM(PS)2 (Rp/Sp) (Rp, Sp-isomers of the bis-phosphorothioateanalog of 2′3′-cGAMP). Examples of TLR agonists include, but are notlimited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR1Oand TLRI 1. Examples of NOD agonists include, but are not limited to,N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyldipeptide (MDP)),gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), anddesmuramylpeptides (DMP).

The “internal transcribed spacer” or “ ITS” is a piece of non-functionalRNA located between structural ribosomal RNAs (rRNA) on a commonprecursor transcript often used for identification of eukaryotic speciesin particular fungi. The rRNA of fungi that forms the core of theribosome is transcribed as a signal gene and consists of the 8S, 5.8Sand 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28Sregions, respectively. These two intercistronic segments between the 18Sand 5.8S and 5.8S and 28S regions are removed by splicing and containsignificant variation between species for barcoding purposes aspreviously described (Schoch et al Nuclear ribosomal internaltranscribed spacer (ITS) region as a universal DNA barcode marker forFungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used forphylogenetic reconstruction however the ITS can serve this function asit is generally highly conserved but contains hypervariable regions thatharbor sufficient nucleotide diversity to differentiate genera andspecies of most fungus.

The term “isolated” or “enriched” encompasses a microbe, EV or otherentity or substance that has been (1) separated from at least some ofthe components with which it was associated when initially produced(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man. Isolatedmicrobes may be separated from at least about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, ormore of the other components with which they were initially associated.In some embodiments, isolated microbes are more than about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified” referto a microbe or other material that has been separated from at leastsome of the components with which it was associated either wheninitially produced or generated (e.g., whether in nature or in anexperimental setting), or during any time after its initial production.A microbe or a microbial population may be considered purified if it isisolated at or after production, such as from a material or environmentcontaining the microbe or microbial population, and a purified microbeor microbial population may contain other materials up to about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or above about 90% and still be considered “isolated.”In some embodiments, purified microbes or microbial population are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. In the instance of microbial compositionsprovided herein, the one or more microbial types present in thecomposition can be independently purified from one or more othermicrobes produced and/or present in the material or environmentcontaining the microbial type. Microbial compositions and the microbialcomponents thereof are generally purified from residual habitatproducts.“

“Metabolite” as used herein refers to any and all molecular compounds,compositions, molecules, ions, co-factors, catalysts or nutrients usedas substrates in any cellular or microbial metabolic reaction orresulting as product compounds, compositions, molecules, ions,co-factors, catalysts or nutrients from any cellular or microbialmetabolic reaction.

“Microbe” refers to any natural or engineered organism characterized asa bacterium, fungus, microscopic alga, protozoan, and the stages ofdevelopment or life cycle stages (e.g., vegetative, spore (includingsporulation, dormancy, and germination), latent, biofilm) associatedwith the organism. Examples of gut microbes include: Actinomycesgraevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila,Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis,Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacteriumadolescentis, Bifidobacterium bifidum, Bilophila wadsworthia, Blautia,Butyrivibrio, Campylobacter gracilis, Clostridia cluster III, Clostridiacluster IV, Clostridia cluster IX (Acidaminococcaceaegroup ), Clostridiacluster XI, Clostridia cluster XIII (Peptostreptococcus group),Clostridia cluster XIV, Clostridia cluster XV, Collinsella aerofaciens,Coprococcus, Corynebacterium sunsvallense, Desulfomonas pigra, Doreaformicigenerans, Dorea longicatena, Escherichia coli, Eubacteriumhadrum, Eubacterium rectale, Faecalibacteria prausnitzii, Gemella,Lactococcus, Lanchnospira, Mollicutes cluster XVI, Mollicutes clusterXVIII, Prevotella, Rothia mucilaginosa, Ruminococcus callidus,Ruminococcus gnavus, Ruminococcus torques, and Streptococcus.

“Microbiome” broadly refers to the microbes residing on or in body siteof a subject or patient. Microbes in a microbiome may include bacteria,viruses, eukaryotic microorganisms, and/or viruses. Individual microbesin a microbiome may be metabolically active, dormant, latent, or existas spores, may exist planktonically or in biofilms, or may be present inthe microbiome in sustainable or transient manner. The microbiome may bea commensal or healthy-state microbiome or a disease-state microbiome.The microbiome may be native to the subject or patient, or components ofthe microbiome may be modulated, introduced, or depleted due to changesin health state (e.g., precancerous or cancerous state) or treatmentconditions (e.g., antibiotic treatment, exposure to different microbes).In some aspects, the microbiome occurs at a mucosal surface. In someaspects, the microbiome is a gut microbiome. In some aspects, themicrobiome is a tumor microbiome.

A “microbiome profile” or a “microbiome signature” of a tissue or samplerefers to an at least partial characterization of the bacterial makeupof a microbiome. In some embodiments, a microbiome profile indicateswhether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strainsare present or absent in a microbiome. In some embodiments, a microbiomeprofile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morecancer-associated bacterial strains are present in a sample. In someembodiments, the microbiome profile indicates the relative or absoluteamount of each bacterial strain detected in the sample. In someembodiments, the microbiome profile is a cancer-associated microbiomeprofile. A cancer-associated microbiome profile is a microbiome profilethat occurs with greater frequency in a subject who has cancer than inthe general population. In some embodiments, the cancer-associatedmicrobiome profile comprises a greater number of or amount ofcancer-associated bacteria than is normally present in a microbiome ofan otherwise equivalent tissue or sample taken from an individual whodoes not have cancer.

“Modified” in reference to a bacteria broadly refers to a bacteria thathas undergone a change from its wild-type form. Examples of bacterialmodifications include genetic modification, gene expression, phenotypemodification, formulation, chemical modification, and dose orconcentration. Examples of improved properties are described throughoutthis specification and include, e.g., attenuation, auxotrophy, homing,or antigenicity. Phenotype modification might include, by way ofexample, bacteria growth in media that modify the phenotype of abacterium that increase or decrease virulence.

As used herein, a gene is “overexpressed” in a bacteria if it isexpressed at a higher level in an engineered bacteria under at leastsome conditions than it is expressed by a wild-type bacteria of the samespecies under the same conditions. Similarly, a gene is “underexpressed”in a bacteria if it is expressed at a lower level in an engineeredbacteria under at least some conditions than it is expressed by awild-type bacteria of the same species under the same conditions.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), micro RNA (miRNA), silencing RNA (siRNA), transferRNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. A polynucleotide may be further modified, such as byconjugation with a labeling component. In all nucleic acid sequencesprovided herein, U nucleotides are interchangeable with T nucleotides.

An “oncobiome” as used herein comprises pathogenic, tumorigenic and/orcancer-associated microbiota, wherein the microbiota comprises one ormore of a virus, a bacterium, a fungus, a protist, a parasite, oranother microbe.

“Oncotrophic” or “oncophilic” microbes and bacteria are microbes thatare highly associated or present in a cancer microenvironment. They maybe preferentially selected for within the environment, preferentiallygrow in a cancer microenvironment or hone to a said environment.

“Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in aphylogenetic tree and is defined by a nucleic acid sequence, e.g., theentire genome, or a specific genetic sequence, and all sequences thatshare sequence identity to this nucleic acid sequence at the level ofspecies. In some embodiments the specific genetic sequence may be the16S sequence or a portion of the 16S sequence. In other embodiments, theentire genomes of two entities are sequenced and compared. In anotherembodiment, select regions such as multilocus sequence tags (MLST),specific genes, or sets of genes may be genetically compared. For 16S,OTUs that share ≥97% average nucleotide identity across the entire 16Sor some variable region of the 16S are considered the same OTU. See e.g.Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ross R P,and O'Toole P W. 2010. Comparison of two next-generation sequencingtechnologies for resolving highly complex microbiota composition usingtandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200.Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterialspecies definition in the genomic era. Philos Trans R Soc Lond B BiolSci 361: 1929-1940. For complete genomes, MLSTs, specific genes, otherthan 16S, or sets of genes OTUs that share ≥95% average nucleotideidentity are considered the same OTU. See e.g., Achtman M, and Wagner M.2008. Microbial diversity and the genetic nature of microbial species.Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, andTiedje J M. 2006. The bacterial species definition in the genomic era.Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequentlydefined by comparing sequences between organisms. Generally, sequenceswith less than 95% sequence identity are not considered to form part ofthe same OTU. OTUs may also be characterized by any combination ofnucleotide markers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof. Operational TaxonomicUnits (OTUs) with taxonomic assignments made to, e.g., genus, species,and phylogenetic clade are provided herein.

As used herein, the term “extracellular vesicle” or “EV” or refers to acomposition derived from a bacteria that comprises bacterial lipids, andbacterial proteins and/or bacterial nucleic acids and/or carbohydratemoieties contained in a nanoparticle. These EVs may contain 1, 2, 3, 4,5, 10, or more than 10 different lipid species. EVs may contain 1, 2, 3,4, 5, 10, or more than 10 different protein species. EVs may contain 1,2, 3, 4, 5, 10, or more than 10 different nucleic acid species. EVs maycontain 1, 2, 3, 4, 5, 10, or more than 10 different carbohydratespecies.

As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified” referto a EV or other material that has been separated from at least some ofthe components with which it was associated either when initiallyproduced or generated (e.g., whether in nature or in an experimentalsetting), or during any time after its initial production. An EV may beconsidered purified if it is isolated at or after production, such asfrom one or more other bacterial components, and a purified microbe ormicrobial population may contain other materials up to about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or above about 90% and still be considered “purified.” Insome embodiments, purified EVs are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. EVcompositions and the microbial components thereof are, e.g., purifiedfrom residual habitat products.

As used herein, the term “purified EV composition” or “EV composition”refer to a preparation that includes EVs that have been separated fromat least one associated substance found in a source material (e.g.separated from at least one other bacterial component) or any materialassociated with the EVs in any process used to produce the preparation.It also refers to a composition that has been significantly enriched orconcentrated. In some embodiments the EVs are concentrated by 2 fold,3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold ormore than 10,000 fold.

“Residual habitat products” refers to material derived from the habitatfor microbiota within or on a subject. For example, microbes live infeces in the gastrointestinal tract, on the skin itself, in saliva,mucus of the respiratory tract, or secretions of the genitourinary tract(i.e., biological matter associated with the microbial community).Substantially free of residual habitat products means that the microbialcomposition no longer contains the biological matter associated with themicrobial environment on or in the human or animal subject and is 100%free, 99% free, 98% free, 97% free, 96% free, or 95% free of anycontaminating biological matter associated with the microbial community.Residual habitat products can include abiotic materials (includingundigested food) or it can include unwanted microorganisms.Substantially free of residual habitat products may also mean that themicrobial composition contains no detectable cells from a human oranimal and that only microbial cells are detectable. In one embodiment,substantially free of residual habitat products may also mean that themicrobial composition contains no detectable viral (including microbialviruses (e.g., phage)), fungal, mycoplasmal contaminants. In anotherembodiment, it means that fewer than 1×10-2%, 1×10-3%, 1×10-4%, 1×10-5%,1×10-6%, 1×10-7%, 1×10-8% of the viable cells in the microbialcomposition are human or animal, as compared to microbial cells. Thereare multiple ways to accomplish this degree of purity, none of which arelimiting. Thus, contamination may be reduced by isolating desiredconstituents through multiple steps of streaking to single colonies onsolid media until replicate (such as, but not limited to, two) streaksfrom serial single colonies have shown only a single colony morphology.Alternatively, reduction of contamination can be accomplished bymultiple rounds of serial dilutions to single desired cells (e.g., adilution of 10-8 or 10-9), such as through multiple 10-fold serialdilutions. This can further be confirmed by showing that multipleisolated colonies have similar cell shapes and Gram staining behavior.Other methods for confirming adequate purity include genetic analysis(e.g., PCR, DNA sequencing), serology and antigen analysis, enzymaticand metabolic analysis, and methods using instrumentation such as flowcytometry with reagents that distinguish desired constituents fromcontaminants.

As used herein, “specific binding” refers to the ability of an antibodyto bind to a predetermined antigen or the ability of a polypeptide tobind to its predetermined binding partner. Typically, an antibody orpolypeptide specifically binds to its predetermined antigen or bindingpartner with an affinity corresponding to a K_(D) of about 10⁻⁷ M orless, and binds to the predetermined antigen/binding partner with anaffinity (as expressed by K_(D)) that is at least 10 fold less, at least100 fold less or at least 1000 fold less than its affinity for bindingto a non-specific and unrelated antigen/binding partner (e.g., BSA,casein). Alternatively, specific binding applies more broadly to a twocomponent system where one component is a protein, lipid, orcarbohydrate or combination thereof and engages with the secondcomponent which is a protein, lipid, carbohydrate or combination thereofin a specific way.

The terms “subject” or “patient” refers to any animal. A subject or apatient described as “in need thereof” refers to one in need of atreatment for a disease. Mammals (i.e., mammalian animals) includehumans, laboratory animals (e.g., primates, rats, mice), livestock(e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats,rodents).

“Strain” refers to a member of a bacterial species with a geneticsignature such that it may be differentiated from closely-relatedmembers of the same bacterial species. The genetic signature may be theabsence of all or part of at least one gene, the absence of all or partof at least on regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the absence (“curing”) of at leastone native plasmid, the presence of at least one recombinant gene, thepresence of at least one mutated gene, the presence of at least oneforeign gene (a gene derived from another species), the presence atleast one mutated regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the presence of at least onenon-native plasmid, the presence of at least one antibiotic resistancecassette, or a combination thereof. Genetic signatures between differentstrains may be identified by PCR amplification optionally followed byDNA sequencing of the genomic region(s) of interest or of the wholegenome. In the case in which one strain (compared with another of thesame species) has gained or lost antibiotic resistance or gained or losta biosynthetic capability (such as an auxotrophic strain), strains maybe differentiated by selection or counter-selection using an antibioticor nutrient/metabolite, respectively.

As used herein, the term “treating” a disease in a subject or “treating”a subject having or suspected of having a disease refers to subjectingthe subject to a pharmaceutical treatment, e.g., the administration ofone or more agents, such that at least one symptom of the disease isdecreased or prevented from worsening. Thus, in one embodiment,“treating” refers inter alia to delaying progression, expeditingremission, inducing remission, augmenting remission, speeding recovery,increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof.

Bacteria

In certain aspects, provided herein are pharmaceutical compositions thatcomprise bacteria and/or EVs made from bacteria.

In some embodiments, the bacteria from which the EVs are obtained aremodified to enhance EV production, to enhance oral delivery of theproduced EVs (e.g., by improving acid resistance, muco-adherence and/orpenetration and/or resistance to bile acids, digestive enzymes,resistance to anti-microbial peptides and/or antibody neutralization),to target desired cell types (e.g. M-cells, goblet cells, enterocytes,dendritic cells, macrophages), to enhance their immunomodulatory and/ortherapeutic effect of the produced EVs (e.g., either alone or incombination with another therapeutic agent), and/or to enhance immuneactivation or suppression by the produced EVs (e.g., through modifiedproduction of polysaccharides, pili, fimbriae, adhesins). In someembodiments, the engineered bacteria described herein are modified toimprove bacterial and/or EV manufacturing (e.g., higher oxygentolerance, stability, improved freeze-thaw tolerance, shorter generationtimes). For example, in some embodiments, the engineered bacteriadescribed include bacteria harboring one or more genetic changes, suchchange being an insertion, deletion, translocation, or substitution, orany combination thereof, of one or more nucleotides contained on thebacterial chromosome or endogenous plasmid and/or one or more foreignplasmids, wherein the genetic change may results in the overexpressionand/or underexpression of one or more genes. The engineered microbe(s)may be produced using any technique known in the art, including but notlimited to site-directed mutagenesis, transposon mutagenesis,knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemicalmutagenesis, ultraviolet light mutagenesis, transformation (chemicallyor by electroporation), phage transduction, directed evolution, or anycombination thereof.

As used herein, the term “bacteria” broadly refers to the domain ofprokaryotic organisms, including Gram positive and Gram negativeorganisms. Examples of species and/or strains of bacteria that can beused to produce the EVs described herein are provided in Tables 1 and/orTable 2 and elsewhere throughout the specification. In some embodiments,the bacterial strain is a bacterial strain having a genome that has atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequenceidentity to a strain listed in Table 1 and/or Table 2. In someembodiments, the EVs are from an oncotrophic bacteria. In someembodiments, the EVs are from an immunostimulatory bacteria. In someembodiments the EVs are from an immunosuppressive bacteria. In someembodiments, the EVs are from an immunomodulatory bacteria. In certainembodiments, EVs are generated from a combination of bacterial strainsprovided herein. In some embodiments, the combination is a combinationof at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,35, 40, 45 or 50 bacterial strains. In some embodiments combinationincludes EVs from bacterial strains listed in Table 1 and/or Table 2and/or bacterial strains having a genome that has at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strainlisted in Table 1 and/or Table 2.

TABLE 1 Exemplary Bacterial Strains Public DB OTU Accession Abiotrophiadefectiva ACIN02000016 Abiotrophia para_adiacens AB022027 Abiotrophiasp. oral clone P4PA_155 P1 AY207063 Acetanaerobacterium elongatumNR_042930 Acetivibrio cellulolyticus NR_025917 Acetivibrioethanolgignens FR749897 Acetobacter aceti NR_026121 Acetobacter fabarumNR_042678 Acetobacter lovaniensis NR_040832 Acetobacter malorumNR_025513 Acetobacter orientalis NR_028625 Acetobacter pasteurianusNR_026107 Acetobacter pomorum NR_042112 Acetobacter syzygii NR_040868Acetobacter tropicalis NR_036881 Acetobacteraceae bacterium AT_5844AGEZ01000040 Acholeplasma laidlawii NR_074448 Achromobacterdenitrificans NR_042021 Achromobacter piechaudii ADMS01000149Achromobacter xylosoxidans ACRC01000072 Acidaminococcus fermentansCP001859 Acidaminococcus intestini CP003058 Acidaminococcus sp. D21ACGB01000071 Acidilobus saccharovorans AY350586 Acidithiobacillusferrivorans NR_074660 Acidovorax sp. 98_63833 AY258065 Acinetobacterbaumannii ACYQ01000014 Acinetobacter calcoaceticus AM157426Acinetobacter genomosp. C1 AY278636 Acinetobacter haemolyticusADMT01000017 Acinetobacter johnsonii ACPL01000162 Acinetobacter juniiACPM01000135 Acinetobacter lwoffii ACPN01000204 Acinetobacter parvusAIEB01000124 Acinetobacter radioresistens ACVR01000010 Acinetobacterschindleri NR_025412 Acinetobacter sp. 56A1 GQ178049 Acinetobacter sp.CIP 101934 JQ638573 Acinetobacter sp. CIP 102143 JQ638578 Acinetobactersp. CIP 53.82 JQ638584 Acinetobacter sp. M16_22 HM366447 Acinetobactersp. RUH2624 ACQF01000094 Acinetobacter sp. SH024 ADCH01000068Actinobacillus actinomycetemcomitans AY362885 Actinobacillus minorACFT01000025 Actinobacillus pleuropneumoniae NR_074857 Actinobacillussuccinogenes CP000746 Actinobacillus ureae AEVG01000167 Actinobaculummassiliae AF487679 Actinobaculum schaalii AY957507 Actinobaculum sp.BM#101342 AY282578 Actinobaculum sp. P2P_19 P1 AY207066 Actinomycescardiffensis GU470888 Actinomyces europaeus NR_026363 Actinomyces funkeiHQ906497 Actinomyces genomosp. C1 AY278610 Actinomyces genomosp. C2AY278611 Actinomyces genomosp. P1 oral clone MB6_C03 DQ003632Actinomyces georgiae GU561319 Actinomyces israelii AF479270 Actinomycesmassiliensis AB545934 Actinomyces meyeri GU561321 Actinomyces naeslundiiX81062 Actinomyces nasicola AJ508455 Actinomyces neuii X71862Actinomyces odontolyticus ACYT01000123 Actinomyces oricola NR_025559Actinomyces orihominis AJ575186 Actinomyces oris BABV01000070Actinomyces sp. 7400942 EU484334 Actinomyces sp. c109 AB16723 9Actinomyces sp. CCUG 37290 AJ234058 Actinomyces sp. ChDC Bl97 AF543275Actinomyces sp. GEJ15 GU561313 Actinomyces sp. HKU31 HQ335393Actinomyces sp. ICM34 HQ616391 Actinomyces sp. ICM41 HQ616392Actinomyces sp. ICM47 HQ616395 Actinomyces sp. ICM54 HQ616398Actinomyces sp. M2231_94_1 AJ234063 Actinomyces sp. oral clone GU009AY349361 Actinomyces sp. oral clone GU067 AY349362 Actinomyces sp. oralclone IO076 AY349363 Actinomyces sp. oral clone IO077 AY349364Actinomyces sp. oral clone IP073 AY349365 Actinomyces sp. oral cloneIP081 AY349366 Actinomyces sp. oral clone JA063 AY349367 Actinomyces sp.oral taxon 170 AFBL01000010 Actinomyces sp. oral taxon 171 AECW01000034Actinomyces sp. oral taxon 178 AEUH01000060 Actinomyces sp. oral taxon180 AEPP01000041 Actinomyces sp. oral taxon 848 ACUY01000072 Actinomycessp. oral taxon C55 HM099646 Actinomyces sp. TeJ5 GU561315 Actinomycesurogenitalis ACFH01000038 Actinomyces viscosus ACRE01000096Adlercreutzia equolifaciens AB306661 Aerococcus sanguinicola AY837833Aerococcus urinae CP002512 Aerococcus urinaeequi NR_043443 Aerococcusviridans ADNT01000041 Aeromicrobium marinum NR_025681 Aeromicrobium sp.JC14 JF824798 Aeromonas allosaccharophila S39232 Aeromonasenteropelogenes X71121 Aeromonas hydrophila NC_008570 Aeromonas jandaeiX60413 Aeromonas salmonicida NC_009348 Aeromonas trota X60415 Aeromonasveronii NR_044845 Afipia genomosp. 4 EU117385 Aggregatibacteractinomycetemcomitans CP001733 Aggregatibacter aphrophilus CP001607Aggregatibacter segnis AEPS01000017 Agrobacterium radiobacter CP000628Agrobacterium tumefaciens AJ3 89893 Agrococcus jenensis NR_026275Akkermansia muciniphila CP001071 Alcaligenes faecalis AB680368Alcaligenes sp. CO14 DQ643040 Alcaligenes sp. S3 HQ262549Alicyclobacillus acidocaldarius NR_074721 Alicyclobacillusacidoterrestris NR_040844 Alicyclobacillus contaminans NR_041475Alicyclobacillus cycloheptanicus NR_024754 Alicyclobacillus herbariusNR_024753 Alicyclobacillus pomorum NR_024801 Alicyclobacillus sp. CCUG53762 HE613268 Alistipes finegoldii NR_043064 Alistipes indistinctusAB490804 Alistipes onderdonkii NR_043318 Alistipes putredinisABFK02000017 Alistipes shahii FP929032 Alistipes sp. HGB5 AENZ01000082Alistipes sp. JC50 JF824804 Alistipes sp. RMA 9912 GQ140629 Alkaliphilusmetalliredigenes AY137848 Alkaliphilus oremlandii NR_043674Alloscardovia omnicolens NR_042583 Alloscardovia sp. OB7196 AB425070Anaerobaculum hydrogeniformans ACJX02000009 Anaerobiospirillumsucciniciproducens NR_026075 Anaerobiospirillum thomasii AJ420985Anaerococcus hydrogenalis ABXA01000039 Anaerococcus lactolyticusABYO01000217 Anaerococcus octavius NR_026360 Anaerococcus prevotiiCP001708 Anaerococcus sp. 8404299 HM587318 Anaerococcus sp. 8405254HM587319 Anaerococcus sp. 9401487 HM587322 Anaerococcus sp. 9403502HM587325 Anaerococcus sp. gpac104 AM176528 Anaerococcus sp. gpac126AM176530 Anaerococcus sp. gpac155 AM176536 Anaerococcus sp. gpac199AM176539 Anaerococcus sp. gpac215 AM176540 Anaerococcus tetradiusACGC01000107 Anaerococcus vaginalis ACXU01000016 Anaerofustisstercorihominis ABIL02000005 Anaeroglobus geminatus AGCJ01000054Anaerosporobacter mobilis NR_042953 Anaerostipes caccae ABAX03000023Anaerostipes sp. 3_2_56FAA ACWB01000002 Anaerotruncus colihominisABGD02000021 Anaplasma marginale ABOR01000019 Anaplasma phagocytophilumNC_007797 Aneurinibacillus aneurinilyticus AB101592 Aneurinibacillusdanicus NR_028657 Aneurinibacillus migulanus NR_036799 Aneurinibacillusterranovensis NR_042271 Aneurinibacillus thermoaerophilus NR_029303Anoxybacillus contaminans NR_029006 Anoxybacillus flavithermus NR_074667Arcanobacterium haemolyticum NR_025347 Arcanobacterium pyogenes GU585578Arcobacter butzleri AEPT01000071 Arcobacter cryaerophilus NR_025905Arthrobacter agilis NR_026198 Arthrobacter arilaitensis NR_074608Arthrobacter bergerei NR_025612 Arthrobacter globiformis NR_026187Arthrobacter nicotianae NR_026190 Atopobium minutum HM007583 Atopobiumparvulum CP001721 Atopobium rimae ACFE01000007 Atopobium sp. BS2HQ616367 Atopobium sp. F0209 EU592966 Atopobium sp. ICM42b10 HQ616393Atopobium sp. ICM57 HQ616400 Atopobium vaginae AEDQ01000024 Aurantimonascoralicida AY065627 Aureimonas altamirensis FN658986 Auritibacterignavus FN554542 Averyella dalhousiensis DQ481464 Bacillus aeoliusNR_025557 Bacillus aerophilus NR_042339 Bacillus aestuarii GQ980243Bacillus alcalophilus X76436 Bacillus amyloliquefaciens NR_075005Bacillus anthracis AAEN01000020 Bacillus atrophaeus NR_075016 Bacillusbadius NR_036893 Bacillus cereus ABDJ01000015 Bacillus circulansAB271747 Bacillus clausii FN397477 Bacillus coagulans DQ297928 Bacillusfirmus NR_025842 Bacillus flexus NR_024691 Bacillus fordii NR_025786Bacillus gelatini NR_025595 Bacillus halmapalus NR_026144 Bacillushalodurans AY144582 Bacillus herbersteinensis NR_042286 Bacillus hortiNR_036860 Bacillus idriensis NR_043268 Bacillus lentus NR_040792Bacillus licheniformis NC_006270 Bacillus megaterium GU252124 Bacillusnealsonii NR_044546 Bacillus niabensis NR_043334 Bacillus niaciniNR_024695 Bacillus pocheonensis NR_041377 Bacillus pumilus NR_074977Bacillus safensis JQ624766 Bacillus simplex NR_042136 Bacillussonorensis NR_025130 Bacillus sp. 10403023 MM10403188 CAET01000089Bacillus sp. 2_A_57_CT2 ACWD01000095 Bacillus sp. 2008724126 GU252108Bacillus sp. 2008724139 GU252111 Bacillus sp. 7_16AIA FN397518 Bacillussp. 9_3AIA FN397519 Bacillus sp. AP8 JX101689 Bacillus sp. B27(2008)EU362173 Bacillus sp. BT1B_CT2 ACWC01000034 Bacillus sp. GB1.1 FJ897765Bacillus sp. GB9 FJ897766 Bacillus sp. HU19.1 FJ897769 Bacillus sp. HU29FJ897771 Bacillus sp. HU33.1 FJ897772 Bacillus sp. JC6 JF824800 Bacillussp. oral taxon F26 HM099642 Bacillus sp. oral taxon F28 HM099650Bacillus sp. oral taxon F79 HM099654 Bacillus sp. SRC_DSF1 GU797283Bacillus sp. SRC_DSF10 GU797292 Bacillus sp. SRC_DSF2 GU797284 Bacillussp. SRC_DSF6 GU797288 Bacillus sp. tc09 HQ844242 Bacillus sp. zh168FJ851424 Bacillus sphaericus DQ286318 Bacillus sporothermoduransNR_026010 Bacillus subtilis EU627588 Bacillus thermoamylovoransNR_029151 Bacillus thuringiensis NC_008600 Bacillus weihenstephanensisNR_074926 Bacteroidales bacterium ph8 JN837494 Bacteroidales genomosp.P1 AY341819 Bacteroidales genomosp. P2 oral clone MB1_G13 DQ003613Bacteroidales genomosp. P3 oral clone MB1_G34 DQ003615 Bacteroidalesgenomosp. P4 oral clone MB2_G17 DQ003617 Bacteroidales genomosp. P5 oralclone MB2_P04 DQ003619 Bacteroidales genomosp. P6 oral clone MB3_C19DQ003634 Bacteroidales genomosp. P7 oral clone MB3_P19 DQ003623Bacteroidales genomosp. P8 oral clone MB4_G15 DQ003626 Bacteroidesacidifaciens NR_028607 Bacteroides barnesiae NR_041446 Bacteroidescaccae EU136686 Bacteroides cellulosilyticus ACCH01000108 Bacteroidesclarus AFBM01000011 Bacteroides coagulans AB547639 Bacteroides coprocolaABIY02000050 Bacteroides coprophilus ACBW01000012 Bacteroides doreiABWZ01000093 Bacteroides eggerthii ACWG01000065 Bacteroides faecisGQ496624 Bacteroides finegoldii AB222699 Bacteroides fluxus AFBN01000029Bacteroides fragilis AP006841 Bacteroides galacturonicus DQ497994Bacteroides helcogenes CP002352 Bacteroides heparinolyticus JN867284Bacteroides intestinalis ABJL02000006 Bacteroides massiliensis AB200226Bacteroides nordii NR_043017 Bacteroides oleiciplenus AB547644Bacteroides ovatus ACWH01000036 Bacteroides pectinophilus ABVQ01000036Bacteroides plebeius AB200218 Bacteroides pyogenes NR_041280 Bacteroidessalanitronis CP002530 Bacteroides salyersiae EU136690 Bacteroides sp.1_1_14 ACRP01000155 Bacteroides sp. 1_1_30 ADCL01000128 Bacteroides sp.1_1_6 ACIC01000215 Bacteroides sp. 2_1_22 ACPQ01000117 Bacteroides sp.2_1_56FAA ACWI01000065 Bacteroides sp. 2_2_4 ABZZ01000168 Bacteroidessp. 20_3 ACRQ01000064 Bacteroides sp. 3_1_19 ADCJ01000062 Bacteroidessp. 3_1_23 ACRS01000081 Bacteroides sp. 3_1_33FAA ACPS01000085Bacteroides sp. 3_1_40A ACRT01000136 Bacteroides sp. 3_2_5 ACIB01000079Bacteroides sp. 315_5 FJ848547 Bacteroides sp. 31SF15 AJ583248Bacteroides sp. 31SF18 AJ583249 Bacteroides sp. 35AE31 AJ583244Bacteroides sp. 35AE37 AJ583245 Bacteroides sp. 35BE34 AJ583246Bacteroides sp. 35BE35 AJ583247 Bacteroides sp. 4_1_36 ACTC01000133Bacteroides sp. 4_3_47FAA ACDR02000029 Bacteroides sp. 9_1_42FAAACAA01000096 Bacteroides sp. AR20 AF139524 Bacteroides sp. AR29 AF139525Bacteroides sp. B2 EU722733 Bacteroides sp. D1 ACAB02000030 Bacteroidessp. D2 ACGA01000077 Bacteroides sp. D20 ACPT01000052 Bacteroides sp. D22ADCK01000151 Bacteroides sp. F_4 AB470322 Bacteroides sp. NB_8 AB117565Bacteroides sp. WH2 AY895180 Bacteroides sp. XB12B AM230648 Bacteroidessp. XB44A AM230649 Bacteroides stercoris ABFZ02000022 Bacteroidesthetaiotaomicron NR_074277 Bacteroides uniforms AB050110 Bacteroidesureolyticus GQ167666 Bacteroides vulgatus CP000139 Bacteroidesxylanisolvens ADKP01000087 Bacteroidetes bacterium oral taxon D27HM099638 Bacteroidetes bacterium oral taxon F31 HM099643 Bacteroidetesbacterium oral taxon F44 HM099649 Bamesiella intestinihominis AB370251Bamesiella viscericola NR_041508 Bartonella bacilliformis NC_008783Bartonella grahamii CP001562 Bartonella henselae NC_005956 Bartonellaquintana BX897700 Bartonella tamiae EF672728 Bartonella washoensisFJ719017 Bdellovibrio sp. MPA AY294215 Bifidobacteriaceae genomosp. C1AY278612 Bifidobacterium adolescentis AAXD02000018 Bifidobacteriumangulatum ABYS02000004 Bifidobacterium animalis CP001606 Bifidobacteriumbifidum ABQP01000027 Bifidobacterium breve CP002743 Bifidobacteriumcatenulatum ABXY01000019 Bifidobacterium dentium CP001750Bifidobacterium gallicum ABXB03000004 Bifidobacterium infantis AY151398Bifidobacterium kashiwanohense AB491757 Bifidobacterium longumABQQ01000041 Bifidobacterium pseudocatenulatum ABXX02000002Bifidobacterium pseudolongum NR_043442 Bifidobacterium scardoviiAJ307005 Bifidobacterium sp. HM2 AB425276 Bifidobacterium sp. HMLN12JF519685 Bifidobacterium sp. M45 HM626176 Bifidobacterium sp. MSX5BHQ616382 Bifidobacterium sp. TM_7 AB218972 Bifidobacterium thermophilumDQ340557 Bifidobacterium urinalis AJ278695 Bilophila wadsworthiaADCP01000166 Bisgaard Taxon AY683487 Bisgaard Taxon AY683489 BisgaardTaxon AY683491 Bisgaard Taxon AY683492 Blastomonas natatoria NR_040824Blautia coccoides AB571656 Blautia glucerasea AB588023 Blautiaglucerasei AB439724 Blautia hansenii ABYU02000037 Blautiahydrogenotrophica ACBZ01000217 Blautia luti AB691576 Blautia productaAB600998 Blautia schinkii NR_026312 Blautia sp. M25 HM626178 Blautiastercoris HM626177 Blautia wexlerae EF036467 Bordetella bronchisepticaNR_025949 Bordetella holmesii AB683187 Bordetella parapertussisNR_025950 Bordetella pertussis BX640418 Borrelia afzelii ABCU01000001Borrelia burgdorferi ABGI01000001 Borrelia crocidurae DQ057990 Borreliaduttonii NC_011229 Borrelia garinii ABJV01000001 Borrelia hermsiiAY597657 Borrelia hispanica DQ057988 Borrelia persica HM161645 Borreliarecurrentis AF107367 Borrelia sp. NE49 AJ224142 Borrelia spielmaniiABKB01000002 Borrelia turicatae NC_008710 Borrelia valaisianaABCY01000002 Brachybacterium alimentarium NR_026269 Brachybacteriumconglomeratum AB537169 Brachybacterium tyrofermentans NR_026272Brachyspira aalborgi FM178386 Brachyspira pilosicoli NR_075069Brachyspira sp. HIS3 FM178387 Brachyspira sp. HIS4 FM178388 Brachyspirasp. HIS5 FM178389 Brevibacillus agri NR_040983 Brevibacillus brevisNR_041524 Brevibacillus centrosporus NR_043414 Brevibacilluschoshinensis NR_040980 Brevibacillus invocatus NR_041836 Brevibacilluslaterosporus NR_037005 Brevibacillus parabrevis NR_040981 Brevibacillusreuszeri NR_040982 Brevibacillus sp. phR JN837488 Brevibacillusthermoruber NR_026514 Brevibacterium aurantiacum NR_044854Brevibacterium casei JF951998 Brevibacterium epidermidis NR_029262Brevibacterium frigoritolerans NR_042639 Brevibacterium linens AJ315491Brevibacterium mcbrellneri ADNU01000076 Brevibacterium paucivoransEU086796 Brevibacterium sanguinis NR_028016 Brevibacterium sp. H15 AB177640 Brevibacterium sp. JC43 JF824806 Brevundimonas subvibrioidesCP002102 Brucella abortus ACBJ01000075 Brucella canis NR_044652 Brucellaceti ACJD01000006 Brucella melitensis AE009462 Brucella microtiNR_042549 Brucella ovis NC_009504 Brucella sp. 83_13 ACBQ01000040Brucella sp. BO1 EU053207 Brucella suis ACBK01000034 Bryantellaformatexigens ACCL02000018 Buchnera aphidicola NR_074609 Bulleidiaextructa ADFR01000011 Burkholderia ambifaria AAUZ01000009 Burkholderiacenocepacia AAEH01000060 Burkholderia cepacia NR_041719 Burkholderiamallei CP000547 Burkholderia multivorans NC_010086 Burkholderiaoklahomensis DQ108388 Burkholderia pseudomallei CP001408 Burkholderiarhizoxinica HQ005410 Burkholderia sp. 383 CP000151 Burkholderiaxenovorans U86373 Burkholderiales bacterium 1_1_47 ADCQ01000066Butyricicoccus pullicaecorum HH793440 Butyricimonas virosa AB443949Butyrivibrio crossotus ABWN01000012 Butyrivibrio fibrisolvens U41172Caldimonas manganoxidans NR_040787 Caminicella sporogenes NR_025485Campylobacter coli AAFL01000004 Campylobacter concisus CP000792Campylobacter curvus NC_009715 Campylobacter fetus ACLG01001177Campylobacter gracilis ACYG01000026 Campylobacter hominis NC_009714Campylobacter jejuni AL139074 Campylobacter lari CP000932 Campylobacterrectus ACFU01000050 Campylobacter showae ACVQ01000030 Campylobacter sp.FOBRC14 HQ616379 Campylobacter sp. FOBRC15 HQ616380 Campylobacter sp.oral clone BB120 AY005038 Campylobacter sputorum NR_044839 Campylobacterupsaliensis AEPU01000040 Candidatus Arthromitus sp. SFB_mouse_YitNR_074460 Candidatus Sulcia muelleri CP002163 Capnocytophaga canimorsusCP002113 Capnocytophaga genomosp. C1 AY278613 Capnocytophaga gingivalisACLQ01000011 Capnocytophaga granulosa X97248 Capnocytophaga ochraceaAEOH01000054 Capnocytophaga sp. GEJ8 GU561335 Capnocytophaga sp. oralclone AH015 AY005074 Capnocytophaga sp. oral clone ASCH05 AY923149Capnocytophaga sp. oral clone ID062 AY349368 Capnocytophaga sp. oralstrain A47ROY AY005077 Capnocytophaga sp. oral strain S3 AY005073Capnocytophaga sp. oral taxon 338 AEXX01000050 Capnocytophaga sp. S1bU42009 Capnocytophaga sputigena ABZV01000054 Cardiobacterium hominisACKY01000036 Cardiobacterium valvarum NR_028847 Camobacterium divergensNR_044706 Camobacterium maltaromaticum NC_019425 Catabacterhongkongensis AB671763 Catenibacterium mitsuokai AB030224 Catonellagenomosp. P1 oral clone MB5_P12 DQ003629 Catonella morbi ACIL02000016Catonella sp. oral clone FL037 AY349369 Cedecea davisae AF493976Cellulosimicrobium funkei AY501364 Cetobacterium somerae AJ438155Chlamydia muridarum AE002160 Chlamydia psittaci NR_036864 Chlamydiatrachomatis U68443 Chlamydiales bacterium NS11 JN606074 Chlamydialesbacterium NS13 JN606075 Chlamydiales bacterium NS16 JN606076Chlamydophila pecorum D88317 Chlamydophila pneumoniae NC_002179Chlamydophila psittaci D85712 Chloroflexi genomosp. P1 AY331414Christensenella minuta AB490809 Chromobacterium violaceum NC_005085Chryseobacterium anthropi AM982793 Chryseobacterium gleum ACKQ02000003Chryseobacterium hominis NR_042517 Citrobacter amalonaticus FR870441Citrobacter braakii NR_028687 Citrobacter farmeri AF025371 Citrobacterfreundii NR_028894 Citrobacter gillenii AF025367 Citrobacter koseriNC_009792 Citrobacter murliniae AF025369 Citrobacter rodentium NR_074903Citrobacter sedlakii AF025364 Citrobacter sp. 30_2 ACDJ01000053Citrobacter sp. KMSI_3 GQ468398 Citrobacter werkmanii AF025373Citrobacter youngae ABWL02000011 Cloacibacillus evryensis GQ258966Clostridiaceae bacterium END_2 EF451053 Clostridiaceae bacterium JC13JF824807 Clostridiales bacterium 1_7_47FAA ABQR01000074 Clostridialesbacterium 9400853 HM587320 Clostridiales bacterium 9403326 HM587324Clostridiales bacterium oral clone P4PA_66 P1 AY207065 Clostridialesbacterium oral taxon 093 GQ422712 Clostridiales bacterium oral taxon F32HM099644 Clostridiales bacterium ph2 JN837487 Clostridiales bacteriumSY8519 AB477431 Clostridiales genomosp. BVAB3 CP001850 Clostridiales sp.SM4_1 FP929060 Clostridiales sp. SS3_4 AY305316 Clostridiales sp. SSC_2FP929061 Clostridium acetobutylicum NR_074511 Clostridium aerotoleransX76163 Clostridium aldenense NR_043680 Clostridium aldrichii NR_026099Clostridium algidicamis NR_041746 Clostridium algidixylanolyticumNR_028726 Clostridium aminovalericum NR_029245 Clostridium amygdalinumAY353957 Clostridium argentinense NR_029232 Clostridium asparagiformeACCJ01000522 Clostridium baratii NR_029229 Clostridium bartlettiiABEZ02000012 Clostridium beijerinckii NR_074434 Clostridium bifermentansX73437 Clostridium bolteae ABCC02000039 Clostridium botulinum NC_010723Clostridium butyricum ABDT01000017 Clostridium cadaveris AB542932Clostridium carboxidivorans FR733710 Clostridium camis NR_044716Clostridium celatum X77844 Clostridium celerecrescens JQ246092Clostridium cellulosi NR_044624 Clostridium chauvoei EU106372Clostridium citroniae ADLJ01000059 Clostridium clariflavum NR_041235Clostridium clostridiiformes M59089 Clostridium clostridioformeNR_044715 Clostridium coccoides EF025906 Clostridium cochleariumNR_044717 Clostridium cocleatum NR_026495 Clostridium colicanis FJ957863Clostridium colinum NR_026151 Clostridium difficile NC_013315Clostridium disporicum NR_026491 Clostridium estertheticum NR_042153Clostridium fallax NR_044714 Clostridium favososporum X76749 Clostridiumfelsineum AF270502 Clostridium frigidicamis NR_024919 Clostridiumgasigenes NR_024945 Clostridium ghonii AB542933 Clostridium glycolicumFJ384385 Clostridium glycyrrhizinilyticum AB233029 Clostridiumhaemolyticum NR_024749 Clostridium hathewayi AY552788 Clostridiumhiranonis AB023970 Clostridium histolyticum HF558362 Clostridiumhylemonae AB023973 Clostridium indolis AF028351 Clostridium innocuumM23732 Clostridium irregulare NR_029249 Clostridium isatidis NR_026347Clostridium kluyveri NR_074165 Clostridium lactatifermentans NR_025651Clostridium lavalense EF564277 Clostridium leptum AJ305238 Clostridiumlimosum FR870444 Clostridium magnum X77835 Clostridium malenominatumFR749893 Clostridium mayombei FR733682 Clostridium methylpentosumACEC01000059 Clostridium nexile X73443 Clostridium novyi NR_074343Clostridium orbiscindens Y18187 Clostridium oroticum FR749922Clostridium paraputrificum AB536771 Clostridium perfringens ABDW01000023Clostridium phytofermentans NR_074652 Clostridium piliforme D14639Clostridium putrefaciens NR_024995 Clostridium quinii NR_026149Clostridium ramosum M23731 Clostridium rectum NR_029271 Clostridiumsaccharogumia DQ100445 Clostridium saccharolyticum CP002109 Clostridiumsardiniense NR_041006 Clostridium sariagoforme NR_026490 Clostridiumscindens AF262238 Clostridium septicum NR_026020 Clostridium sordelliiAB448946 Clostridium sp. 7_2_43FAA ACDK01000101 Clostridium sp. D5ADBG01000142 Clostridium sp. HGF2 AENW01000022 Clostridium sp. HPB_46AY862516 Clostridium sp. JC122 CAEV01000127 Clostridium sp. L2_50AAYW02000018 Clostridium sp. LMG 16094 X95274 Clostridium sp. M62_1ACFX02000046 Clostridium sp. MLG055 AF304435 Clostridium sp. MT4 EFJ159523 Clostridium sp. NMBHI_1 JN093130 Clostridium sp. NML 04A032EU815224 Clostridium sp. SS2_1 ABGC03000041 Clostridium sp. SY8519AP012212 Clostridium sp. TM_40 AB249652 Clostridium sp. YIT 12069AB491207 Clostridium sp. YIT 12070 AB491208 Clostridium sphenoidesX73449 Clostridium spiroforme X73441 Clostridium sporogenes ABKW02000003Clostridium sporosphaeroides NR_044835 Clostridium stercorariumNR_025100 Clostridium sticklandii L04167 Clostridium straminisolvensNR_024829 Clostridium subterminale NR_041795 Clostridium sulfidigenesNR_044161 Clostridium symbiosum ADLQ01000114 Clostridium tertium Y18174Clostridium tetani NC_004557 Clostridium thermocellum NR_074629Clostridium tyrobutyricum NR_044718 Clostridium viride NR_026204Clostridium xylanolyticum NR_037068 Collinsella aerofaciens AAVN02000007Collinsella intestinalis ABXH02000037 Collinsella stercoris ABXJ01000150Collinsella tanakaei AB490807 Comamonadaceae bacterium NML000135JN585335 Comamonadaceae bacterium NML790751 JN585331 Comamonadaceaebacterium NML910035 JN585332 Comamonadaceae bacterium NML910036 JN585333Comamonadaceae bacterium oral taxon F47 HM099651 Comamonas sp. NSP5AB076850 Conchiformibius kuhniae NR_041821 Coprobacillus cateniformisAB030218 Coprobacillus sp. 29_1 ADKX01000057 Coprobacillus sp. D7ACDT01000199 Coprococcus catus EU266552 Coprococcus comes ABVR01000038Coprococcus eutactus EF031543 Coprococcus sp. ART55_1 AY350746Coriobacteriaceae bacterium BV3Ac1 JN809768 Coriobacteriaceae bacteriumJC110 CAEM01000062 Coriobacteriaceae bacterium phI JN837493Corynebacterium accolens ACGD01000048 Corynebacterium ammoniagenesADNS01000011 Corynebacterium amycolatum ABZU01000033 Corynebacteriumappendicis NR_028951 Corynebacterium argentoratense EF463055Corynebacterium atypicum NR_025540 Corynebacterium aurimucosumACLH01000041 Corynebacterium bovis AF537590 Corynebacterium canisGQ871934 Corynebacterium casei NR_025101 Corynebacterium confusum Y15886Corynebacterium coyleae X96497 Corynebacterium diphtheriae NC_002935Corynebacterium durum Z97069 Corynebacterium efficiens ACLI01000121Corynebacterium falsenii Y13024 Corynebacterium flavescens NR_037040Corynebacterium genitalium ACLJ01000031 Corynebacterium glaucumNR_028971 Corynebacterium glucuronolyticum ABYP01000081 Corynebacteriumglutamicum BA000036 Corynebacterium hansenii AM946639 Corynebacteriumimitans AF537597 Corynebacterium jeikeium ACYW01000001 Corynebacteriumkroppenstedtii NR_026380 Corynebacterium lipophiloflavum ACHJ01000075Corynebacterium macginleyi AB359393 Corynebacterium mastitidis AB359395Corynebacterium matruchotii ACSH02000003 Corynebacterium minutissimumX82064 Corynebacterium mucifaciens NR_026396 Corynebacterium propinquumNR_037038 Corynebacterium pseudodiphtheriticum X84258 Corynebacteriumpseudogenitalium ABYQ01000237 Corynebacterium pseudotuberculosisNR_037070 Corynebacterium pyruviciproducens FJ185225 Corynebacteriumrenale NR_037069 Corynebacterium resistens ADGN01000058 Corynebacteriumriegelii EU848548 Corynebacterium simulans AF537604 Corynebacteriumsingulare NR_026394 Corynebacterium sp. 1 ex sheep Y13427Corynebacterium sp. L_2012475 HE575405 Corynebacterium sp. NML 93_0481GU238409 Corynebacterium sp. NML 97_0186 GU238411 Corynebacterium sp.NML 99_0018 GU238413 Corynebacterium striatum ACGE01000001Corynebacterium sundsvallense Y09655 Corynebacterium tuberculostearicumACVP01000009 Corynebacterium tuscaniae AY677186 Corynebacterium ulceransNR_074467 Corynebacterium urealyticum X81913 Corynebacteriumureicelerivorans AM397636 Corynebacterium variabile NR_025314Corynebacterium xerosis FN179330 Coxiella burnetii CP000890 Cronobactermalonaticus GU122174 Cronobacter sakazakii NC_009778 Cronobacterturicensis FN543093 Cryptobacterium curium GQ422741 Cupriavidusmetallidurans GU230889 Cytophaga xylanolytica FR733683 Deferribacteressp. oral clone JV001 AY349370 Deferribacteres sp. oral clone JV006AY349371 Deferribacteres sp. oral clone JV023 AY349372 Deinococcusradiodurans AE000513 Deinococcus sp. R_43890 FR682752 Delftiaacidovorans CP000884 Dermabacter hominis FJ263375 Dermacoccus sp.Ellin185 AEIQ01000090 Desmospora activa AM940019 Desmospora sp. 8437AFHT01000143 Desulfitobacterium frappieri AJ276701 Desulfitobacteriumhafniense NR_074996 Desulfobulbus sp. oral clone CH031 AY005036Desulfotomaculum nigrificans NR_044832 Desulfovibrio desulfuricansDQ092636 Desulfovibrio fairfieldensis U42221 Desulfovibrio pigerAF192152 Desulfovibrio sp. 3_1_syn3 ADDR01000239 Desulfovibrio vulgarisNR_074897 Dialister invisus ACIM02000001 Dialister micraerophilusAFBB01000028 Dialister microaerophilus AENT01000008 Dialisterpneumosintes HM596297 Dialister propionicifaciens NR_043231 Dialistersp. oral taxon 502 GQ422739 Dialister succinatiphilus AB370249 Dietzianatronolimnaea GQ870426 Dietzia sp. BBDP51 DQ337512 Dietzia sp. CA149GQ870422 Dietzia timorensis GQ870424 Dorea formicigenerans AAXA02000006Dorea longicatena AJ132842 Dysgonomonas gadei ADLV01000001 Dysgonomonasmossii ADLW01000023 Edwardsiella tarda CP002154 Eggerthella lentaAF292375 Eggerthella sinensis AY321958 Eggerthella sp. 1_3_56FAAACWN01000099 Eggerthella sp. HGA1 AEXR01000021 Eggerthella sp. YY7918AP012211 Ehrlichia chaffeensis AAIF01000035 Eikenella corrodensACEA01000028 Enhydrobacter aerosaccus ACYI01000081 Enterobacteraerogenes AJ251468 Enterobacter asburiae NR_024640 Enterobactercancerogenus Z96078 Enterobacter cloacae FP929040 Enterobacter cowaniiNR_025566 Enterobacter hormaechei AFHR01000079 Enterobacter sp. 247BMCHQ122932 Enterobacter sp. 638 NR_074777 Enterobacter sp. JC163 JN657217Enterobacter sp. SCSS HM007811 Enterobacter sp. TSE38 HM156134Enterobacteriaceae bacterium 9_2_54FAA ADCU01000033 Enterobacteriaceaebacterium CF01Ent_1 AJ489826 Enterobacteriaceae bacterium Smarlab3302238 AY538694 Enterococcus avium AF133535 Enterococcus caccaeAY943820 Enterococcus casseliflavus AEWT01000047 Enterococcus duransAJ276354 Enterococcus faecalis AE016830 Enterococcus faecium AM157434Enterococcus gallinarum AB269767 Enterococcus gilvus AY033814Enterococcus hawaiiensis AY321377 Enterococcus hirae AF061011Enterococcus italicus AEPV01000109 Enterococcus mundtii NR_024906Enterococcus raffinosus FN600541 Enterococcus sp. BV2CASA2 JN809766Enterococcus sp. CCRI_16620 GU457263 Enterococcus sp. F95 FJ463817Enterococcus sp. RfL6 AJ133478 Enterococcus thailandicus AY321376Eremococcus coleocola AENN01000008 Erysipelothrix inopinata NR_025594Erysipelothrix rhusiopathiae ACLK01000021 Erysipelothrix tonsillarumNR_040871 Erysipelotrichaceae bacterium 3_1_53 ACTJ01000113Erysipelotrichaceae bacterium 5_2_54FAA ACZW01000054 Escherichiaalbertii ABKX01000012 Escherichia coli NC_008563 Escherichia fergusoniiCU928158 Escherichia hermannii HQ407266 Escherichia sp. 1_1_43ACID0100003 3 Escherichia sp. 4_1_40B ACDM02000056 Escherichia sp. B4EU722735 Escherichia vulneris NR_041927 Ethanoligenens harbinenseAY675965 Eubacteriaceae bacterium P4P_50 P4 AY207060 Eubacterium barkeriNR_044661 Eubacterium biforme ABYT01000002 Eubacterium brachy U13038Eubacterium budayi NR_024682 Eubacterium callanderi NR_026330Eubacterium cellulosolvens AY178842 Eubacterium contortum FR749946Eubacterium coprostanoligenes HM037995 Eubacterium cylindroides FP929041Eubacterium desmolans NR_044644 Eubacterium dolichum L34682 Eubacteriumeligens CP001104 Eubacterium fissicatena FR749935 Eubacterium hadrumFR749933 Eubacterium hallii L34621 Eubacterium infirmum U13039Eubacterium limosum CP002273 Eubacterium moniliforme HF558373Eubacterium multiforme NR_024683 Eubacterium nitritogenes NR_024684Eubacterium nodatum U13041 Eubacterium ramulus AJ011522 Eubacteriumrectale FP929042 Eubacterium ruminantium NR_024661 Eubacterium saburreumAB525414 Eubacterium saphenum NR_026031 Eubacterium siraeum ABCA03000054Eubacterium sp. 3_1_31 ACTL01000045 Eubacterium sp. AS15b HQ616364Eubacterium sp. OBRC9 HQ616354 Eubacterium sp. oral clone GI038 AY349374Eubacterium sp. oral clone IR009 AY349376 Eubacterium sp. oral cloneJH012 AY349373 Eubacterium sp. oral clone JI012 AY349379 Eubacterium sp.oral clone JN088 AY349377 Eubacterium sp. oral clone JS001 AY349378Eubacterium sp. oral clone OH3A AY947497 Eubacterium sp. WAL 14571FJ687606 Eubacterium tenue M59118 Eubacterium tortuosum NR_044648Eubacterium ventriosum L34421 Eubacterium xylanophilum L34628Eubacterium yurii AEES01000073 Ewingella americana JN175329Exiguobacterium acetylicum FJ970034 Facklamia hominis Y10772Faecalibacterium prausnitzii ACOP02000011 Filifactor alocis CP002390Filifactor villosus NR_041928 Finegoldia magna ACHM02000001Flavobacteriaceae genomosp. C1 AY278614 Flavobacterium sp. NF2_1FJ195988 Flavonifractor plautii AY724678 Flexispira rappini AY126479Flexistipes sinusarabici NR_074881 Francisella novicida ABSS01000002Francisella philomiragia AY928394 Francisella tularensis ABAZ01000082Fulvimonas sp. NML 060897 EF589680 Fusobacterium canifelinum AY162222Fusobacterium genomosp. C1 AY278616 Fusobacterium genomosp. C2 AY278617Fusobacterium gonidiaformans ACET01000043 Fusobacterium mortiferumACDB02000034 Fusobacterium naviforme HQ223106 Fusobacterium necrogenesX55408 Fusobacterium necrophorum AM905356 Fusobacterium nucleatumADVK01000034 Fusobacterium periodonticum ACJY01000002 Fusobacteriumrussii NR_044687 Fusobacterium sp. 1_1_41FAA ADGG01000053 Fusobacteriumsp. 11_3_2 ACUO01000052 Fusobacterium sp. 12_1B AGWJ01000070Fusobacterium sp. 2_1_31 ACDC02000018 Fusobacterium sp. 3_1_27ADGF01000045 Fusobacterium sp. 3_1_33 ACQE01000178 Fusobacterium sp.3_1_36A2 ACPU01000044 Fusobacterium sp. 3_1_5R ACDD01000078Fusobacterium sp. AC18 HQ616357 Fusobacterium sp. ACB2 HQ616358Fusobacterium sp. AS2 HQ616361 Fusobacterium sp. CM1 HQ616371Fusobacterium sp. CM21 HQ616375 Fusobacterium sp. CM22 HQ616376Fusobacterium sp. D12 ACDG02000036 Fusobacterium sp. oral clone ASCF06AY923141 Fusobacterium sp. oral clone ASCF11 AY953256 Fusobacteriumulcerans ACDH01000090 Fusobacterium varium ACIE01000009 Gardnerellavaginalis CP001849 Gemella haemolysans ACDZ02000012 Gemella morbillorumNR_025904 Gemella morbillorum ACRX01000010 Gemella sanguinisACRY01000057 Gemella sp. oral clone ASCE02 AY923133 Gemella sp. oralclone ASCF04 AY923139 Gemella sp. oral clone ASCF12 AY923143 Gemella sp.WAL 1945J EU427463 Gemmiger formicilis GU562446 Geobacillus kaustophilusNR_074989 Geobacillus sp. E263 DQ647387 Geobacillus sp. WCH70 CP001638Geobacillus stearothermophilus NR_040794 Geobacillus thermocatenulatusNR_043020 Geobacillus thermodenitrificans NR_074976 Geobacillusthermoglucosidasius NR_043022 Geobacillus thermoleovorans NR_074931Geobacter bemidjiensis CP001124 Gloeobacter violaceus NR_074282Gluconacetobacter azotocaptans NR_028767 Gluconacetobacterdiazotrophicus NR_074292 Gluconacetobacter entanii NR_028909Gluconacetobacter europaeus NR_026513 Gluconacetobacter hanseniiNR_026133 Gluconacetobacter johannae NR_024959 Gluconacetobacteroboediens NR_041295 Gluconacetobacter xylinus NR_074338 Gordoniabronchialis NR_027594 Gordonia polyisoprenivorans DQ385609 Gordonia sp.KTR9 DQ068383 Gordonia sputi FJ536304 Gordonia terrae GQ848239Gordonibacter pamelaeae AM886059 Gordonibacter pamelaeae FP929047Gracilibacter thermotolerans NR_043559 Gramella forsetii NR_074707Granulicatella adiacens ACKZ01000002 Granulicatella elegans AB252689Granulicatella paradiacens AY879298 Granulicatella sp. M658_99_3AJ271861 Granulicatella sp. oral clone ASC02 AY923126 Granulicatella sp.oral clone ASCA05 DQ341469 Granulicatella sp. oral clone ASCB09 AY953251Granulicatella sp. oral clone ASCG05 AY923146 Grimontia hollisaeADAQ01000013 Haematobacter sp. BC14248 GU396991 Haemophilus aegyptiusAFBC01000053 Haemophilus ducreyi AE017143 Haemophilus genomosp. P2 oralclone MB3_C24 DQ003621 Haemophilus genomosp. P3 oral clone MB3_C38DQ003635 Haemophilus haemolyticus JN175335 Haemophilus influenzaeAADP01000001 Haemophilus parahaemolyticus GU561425 Haemophilusparainfluenzae AEWU01000024 Haemophilus paraphrophaemolyticus M75076Haemophilus parasuis GU226366 Haemophilus somnus NC_008309 Haemophilussp. 70334 HQ680854 Haemophilus sp. HK445 FJ685624 Haemophilus sp. oralclone ASCA07 AY923117 Haemophilus sp. oral clone ASCG06 AY923147Haemophilus sp. oral clone BJ021 AY005034 Haemophilus sp. oral cloneBJ095 AY005033 Haemophilus sp. oral clone JM053 AY349380 Haemophilus sp.oral taxon 851 AGRK01000004 Haemophilus sputorum AFNK01000005 Hafniaalvei DQ412565 Halomonas elongata NR_074782 Halomonas johnsoniaeFR775979 Halorubrum lipolyticum AB477978 Helicobacter bilis ACDN01000023Helicobacter canadensis ABQS01000108 Helicobacter cinaedi ABQT01000054Helicobacter pullorum ABQU01000097 Helicobacter pylori CP000012Helicobacter sp. None U44756 Helicobacter winghamensis ACDO01000013Heliobacterium modesticaldum NR_074517 Herbaspirillum seropedicaeCP002039 Herbaspirillum sp. JC206 JN657219 Histophilus somni AF549387Holdemania filiformis Y11466 Hydrogenoanaerobacterium saccharovoransNR_044425 Hyperthermus butylicus CP000493 Hyphomicrobium sulfonivoransAY468372 Hyphomonas neptunium NR_074092 Ignatzschineria indica HQ823562Ignatzschineria sp. NML 95_0260 HQ823559 Ignicoccus islandicus X99562Inquilinus limosus NR_029046 Janibacter limosus NR_026362 Janibactermelonis EF063716 Janthinobacterium sp. SY12 EF455530 Johnsonella ignavaX87152 Jonquetella anthropi ACOO02000004 Kerstersia gyiorum NR_025669Kingella denitrificans AEWV01000047 Kingella genomosp. P1 oral coneMB2_C20 DQ003616 Kingella kingae AFHS01000073 Kingella oralisACJW02000005 Kingella sp. oral clone ID059 AY349381 Klebsiella oxytocaAY292871 Klebsiella pneumoniae CP000647 Klebsiella sp. AS10 HQ616362Klebsiella sp. Co9935 DQ068764 Klebsiella sp. enrichment culture cloneSRC_DSD25 HM195210 Klebsiella sp. OBRC7 HQ616353 Klebsiella sp. SP_BAFJ999767 Klebsiella sp. SRC_DSD1 GU797254 Klebsiella sp. SRC_DSD11GU797263 Klebsiella sp. SRC_DSD12 GU797264 Klebsiella sp. SRC_DSD15GU797267 Klebsiella sp. SRC_DSD2 GU797253 Klebsiella sp. SRC_DSD6GU797258 Klebsiella variicola CP001891 Kluyvera ascorbata NR_028677Kluyvera cryocrescens NR_028803 Kocuria marina GQ260086 Kocuriapalustris EU333884 Kocuria rhizophila AY030315 Kocuria rosea X87756Kocuria varians AF542074 Lachnobacterium bovis GU324407 Lachnospiramultipara FR733699 Lachnospira pectinoschiza L14675 Lachnospiraceaebacterium 1_1_57FAA ACTM01000065 Lachnospiraceae bacterium 1_4_56FAAACTN01000028 Lachnospiraceae bacterium 2_1_46FAA ADLB01000035Lachnospiraceae bacterium 2_1_58FAA ACTO01000052 Lachnospiraceaebacterium 3_1_57FAA_CT1 ACTP01000124 Lachnospiraceae bacterium 4_1_37FAAADCR01000030 Lachnospiraceae bacterium 5_1_57FAA ACTR01000020Lachnospiraceae bacterium 5_1_63FAA ACTS01000081 Lachnospiraceaebacterium 6_1_63FAA ACTV01000014 Lachnospiraceae bacterium 8_1_57FAAACWQ01000079 Lachnospiraceae bacterium 9_1_43BFAA ACTX01000023Lachnospiraceae bacterium A4 DQ789118 Lachnospiraceae bacterium DJF VP30EU728771 Lachnospiraceae bacterium ICM62 HQ616401 Lachnospiraceaebacterium MSX33 HQ616384 Lachnospiraceae bacterium oral taxon 107ADDS01000069 Lachnospiraceae bacterium oral taxon F15 HM099641Lachnospiraceae genomosp. C1 AY278618 Lactobacillus acidipiscisNR_024718 Lactobacillus acidophilus CP000033 Lactobacillus alimentariusNR_044701 Lactobacillus amylolyticus ADNY01000006 Lactobacillusamylovorus CP002338 Lactobacillus antri ACLL01000037 Lactobacillusbrevis EU194349 Lactobacillus buchneri ACGH01000101 Lactobacillus caseiCP000423 Lactobacillus catenaformis M23729 Lactobacillus coleohominisACOH01000030 Lactobacillus coryniformis NR_044705 Lactobacilluscrispatus ACOG01000151 Lactobacillus curvatus NR_042437 Lactobacillusdelbrueckii CP002341 Lactobacillus dextrinicus NR_036861 Lactobacillusfarciminis NR_044707 Lactobacillus fermentum CP002033 Lactobacillusgasseri ACOZ01000018 Lactobacillus gastricus AICN01000060 Lactobacillusgenomosp. C1 AY278619 Lactobacillus genomosp. C2 AY278620 Lactobacillushelveticus ACLM01000202 Lactobacillus hilgardii ACGP01000200Lactobacillus hominis FR681902 Lactobacillus iners AEKJ01000002Lactobacillus jensenii ACQD01000066 Lactobacillus johnsonii AE017198Lactobacillus kalixensis NR_029083 Lactobacillus kefiranofaciensNR_042440 Lactobacillus kefiri NR_042230 Lactobacillus kimchii NR_025045Lactobacillus leichmannii JX986966 Lactobacillus mucosae FR693800Lactobacillus murinus NR_042231 Lactobacillus nodensis NR_041629Lactobacillus oeni NR_043095 Lactobacillus oris AEKL01000077Lactobacillus parabrevis NR_042456 Lactobacillus parabuchneri NR_041294Lactobacillus paracasei ABQV01000067 Lactobacillus parakefiri NR_029039Lactobacillus pentosus JN813103 Lactobacillus perolens NR_029360Lactobacillus plantarum ACGZ02000033 Lactobacillus pontis HM218420Lactobacillus reuteri ACGW02000012 Lactobacillus rhamnosus ABWJ01000068Lactobacillus rogosae GU269544 Lactobacillus ruminis ACGS02000043Lactobacillus sakei DQ989236 Lactobacillus salivarius AEBA01000145Lactobacillus saniviri AB602569 Lactobacillus senioris AB602570Lactobacillus sp. 66c FR681900 Lactobacillus sp. BT6 HQ616370Lactobacillus sp. KLDS 1.0701 EU600905 Lactobacillus sp. KLDS 1.0702EU600906 Lactobacillus sp. KLDS 1.0703 EU600907 Lactobacillus sp. KLDS1.0704 EU600908 Lactobacillus sp. KLDS 1.0705 EU600909 Lactobacillus sp.KLDS 1.0707 EU600911 Lactobacillus sp. KLDS 1.0709 EU600913Lactobacillus sp. KLDS 1.0711 EU600915 Lactobacillus sp. KLDS 1.0712EU600916 Lactobacillus sp. KLDS 1.0713 EU600917 Lactobacillus sp. KLDS1.0716 EU600921 Lactobacillus sp. KLDS 1.0718 EU600922 Lactobacillus sp.KLDS 1.0719 EU600923 Lactobacillus sp. oral clone HT002 AY349382Lactobacillus sp. oral clone HT070 AY349383 Lactobacillus sp. oral taxon052 GQ422710 Lactobacillus tucceti NR_042194 Lactobacillus ultunensisACGU01000081 Lactobacillus vaginalis ACGV01000168 Lactobacillus viniNR_042196 Lactobacillus vitulinus NR_041305 Lactobacillus zeae NR_037122Lactococcus garvieae AF061005 Lactococcus lactis CP002365 Lactococcusraffinolactis NR_044359 Lactonifactor longoviformis DQ100449 Laribacterhongkongensis CP001154 Lautropia mirabilis AEQP01000026 Lautropia sp.oral clone AP009 AY005030 Legionella hackeliae M36028 Legionellalongbeachae M36029 Legionella pneumophila NC_002942 Legionella sp. D3923JN380999 Legionella sp. D4088 JN381012 Legionella sp. H63 JF831047Legionella sp. NML 93L054 GU062706 Legionella steelei HQ398202Leminorella grimontii AJ233421 Leminorella richardii HF558368 Leptospiraborgpetersenii NC_008508 Leptospira broomii NR_043200 Leptospirainterrogans NC_005823 Leptospira licerasiae EF612284 Leptotrichiabuccalis CP001685 Leptotrichia genomosp. C1 AY278621 Leptotrichiagoodfellowii ADAD01000110 Leptotrichia hofstadii ACVB02000032Leptotrichia shahii AY029806 Leptotrichia sp. neutropenicPatientAF189244 Leptotrichia sp. oral clone GT018 AY349384 Leptotrichia sp.oral clone GT020 AY349385 Leptotrichia sp. oral clone HE012 AY349386Leptotrichia sp. oral clone IK040 AY349387 Leptotrichia sp. oral cloneP2PB_51 P1 AY207053 Leptotrichia sp. oral taxon 223 GU408547 Leuconostoccarnosum NR_040811 Leuconostoc citreum AM157444 Leuconostocgasicomitatum FN822744 Leuconostoc inhae NR_025204 Leuconostoc kimchiiNR_075014 Leuconostoc lactis NR_040823 Leuconostoc mesenteroidesACKV01000113 Leuconostoc pseudomesenteroides NR_040814 Listeria grayiACCR02000003 Listeria innocua JF967625 Listeria ivanovii X56151 Listeriamonocytogenes CP002003 Listeria welshimeri AM263198 Luteococcussanguinis NR_025507 Lutispora thermophila NR_041236 Lysinibacillusfusiformis FN397522 Lysinibacillus sphaericus NR_074883 Macrococcuscaseolyticus NR_074941 Mannheimia haemolytica ACZX01000102Marvinbryantia formatexigens AJ505973 Massilia sp. CCUG 43427A FR773700Megamonas funiformis AB300988 Megamonas hypermegale AJ420107 Megasphaeraelsdenii AY038996 Megasphaera genomosp. C1 AY278622 Megasphaeragenomosp. type_1 ADGP01000010 Megasphaera micronuciformis AECS01000020Megasphaera sp. BLPYG_07 HM990964 Megasphaera sp. UPII 199_6AFIJ01000040 Metallosphaera sedula D26491 Methanobacterium formicicumNR_025028 Methanobrevibacter acididurans NR_028779 Methanobrevibacterarboriphilus NR_042783 Methanobrevibacter curvatus NR_044796Methanobrevibacter cuticularis NR_044776 Methanobrevibacter filiformisNR_044801 Methanobrevibacter gottschalkii NR_044789 Methanobrevibactermillerae NR_042785 Methanobrevibacter olleyae NR_043024Methanobrevibacter oralis HE654003 Methanobrevibacter ruminantiumNR_042784 Methanobrevibacter smithii ABYV02000002 Methanobrevibacterthaueri NR_044787 Methanobrevibacter woesei NR_044788 Methanobrevibacterwolinii NR_044790 Methanosphaera stadtmanae AY196684 Methylobacteriumextorquens NC_010172 Methylobacterium podarium AY468363 Methylobacteriumradiotolerans GU294320 Methylobacterium sp. 1sub AY468371Methylobacterium sp. MM4 AY468370 Methylocella silvestris NR_074237Methylophilus sp. ECd5 AY436794 Microbacterium chocolatum NR_037045Microbacterium flavescens EU714363 Microbacterium gubbeenense NR_025098Microbacterium lacticum EU714351 Microbacterium oleivorans EU714381Microbacterium oxydans EU714348 Microbacterium paraoxydans AJ491806Microbacterium phyllosphaerae EU714359 Microbacterium schleiferiNR_044936 Microbacterium sp. 768 EU714378 Microbacterium sp. oral strainC24KA AF287752 Microbacterium testaceum EU714365 Micrococcus antarcticusNR_025285 Micrococcus luteus NR_075062 Micrococcus lylae NR_026200Micrococcus sp. 185 EU714334 Microcystis aeruginosa NC_010296Mitsuokella jalaludinii NR_028840 Mitsuokella multacida ABWK02000005Mitsuokella sp. oral taxon 521 GU413658 Mitsuokella sp. oral taxon G68GU432166 Mobiluncus curtisii AEPZ01000013 Mobiluncus mulierisACKW01000035 Moellerella wisconsensis JN175344 Mogibacterium diversumNR_027191 Mogibacterium neglectum NR_027203 Mogibacterium pumilumNR_028608 Mogibacterium timidum Z36296 Mollicutes bacterium pACH93AY297808 Moorella thermoacetica NR_075001 Moraxella catarrhalis CP002005Moraxella lincolnii FR822735 Moraxella osloensis JN175341 Moraxella sp.16285 JF682466 Moraxella sp. GM2 JF837191 Morganella morganii AJ301681Morganella sp. JB_T16 AJ781005 Morococcus cerebrosus JN175352 Moryellaindoligenes AF527773 Mycobacterium abscessus AGQU01000002 Mycobacteriumafricanum AF480605 Mycobacterium alsiensis AJ938169 Mycobacterium aviumCP000479 Mycobacterium chelonae AB548610 Mycobacterium colombienseAM062764 Mycobacterium elephantis AF385898 Mycobacterium gordonaeGU142930 Mycobacterium intracellulare GQ153276 Mycobacterium kansasiiAF480601 Mycobacterium lacus NR_025175 Mycobacterium leprae FM211192Mycobacterium lepromatosis EU203590 Mycobacterium mageritense FR798914Mycobacterium mantenii FJ042897 Mycobacterium marinum NC_010612Mycobacterium microti NR_025234 Mycobacterium neoaurum AF268445Mycobacterium parascrofulaceum ADNV01000350 Mycobacterium paraterraeEU919229 Mycobacterium phlei GU142920 Mycobacterium seoulense DQ536403Mycobacterium smegmatis CP000480 Mycobacterium sp. 1761 EU703150Mycobacterium sp. 1776 EU703152 Mycobacterium sp. 1781 EU703147Mycobacterium sp. 1791 EU703148 Mycobacterium sp. 1797 EU703149Mycobacterium sp. AQ1GA4 HM210417 Mycobacterium sp. B10_07.09.0206HQ174245 Mycobacterium sp. GN_10546 FJ497243 Mycobacterium sp. GN_10827FJ497247 Mycobacterium sp. GN_11124 FJ652846 Mycobacterium sp. GN_9188FJ497240 Mycobacterium sp. GR_2007_210 FJ555538 Mycobacterium sp. HE5AJ012738 Mycobacterium sp. NLA001000736 HM627011 Mycobacterium sp. WDQ437715 Mycobacterium tuberculosis CP001658 Mycobacterium ulceransAB548725 Mycobacterium vulneris EU834055 Mycoplasma agalactiae AF010477Mycoplasma amphoriforme AY531656 Mycoplasma arthritidis NC_011025Mycoplasma bovoculi NR_025987 Mycoplasma faucium NR_024983 Mycoplasmafermentans CP002458 Mycoplasma flocculare X62699 Mycoplasma genitaliumL43967 Mycoplasma hominis AF443616 Mycoplasma orale AY796060 Mycoplasmaovipneumoniae NR_025989 Mycoplasma penetrans NC_004432 Mycoplasmapneumoniae NC_000912 Mycoplasma putrefaciens U26055 Mycoplasmasalivarium M24661 Mycoplasmataceae genomosp. P1 oral clone DQ003614MB1_G23 Myroides odoratimimus NR_042354 Myroides sp. MY15 GU253339Neisseria bacilliformis AFAY01000058 Neisseria cinerea ACDY01000037Neisseria elongata ADBF01000003 Neisseria flavescens ACQV01000025Neisseria genomosp. P2 oral clone MB5_P15 DQ003630 Neisseria gonorrhoeaeCP002440 Neisseria lactamica ACEQ01000095 Neisseria macacae AFQE01000146Neisseria meningitidis NC_003112 Neisseria mucosa ACDX01000110 Neisseriapharyngis AJ239281 Neisseria polysaccharea ADBE01000137 Neisseria siccaACKO02000016 Neisseria sp. KEM232 GQ203291 Neisseria sp. oral cloneAPI32 AY005027 Neisseria sp. oral clone JC012 AY349388 Neisseria sp.oral strain B33KA AY005028 Neisseria sp. oral taxon 014 ADEA01000039Neisseria sp. SMC_A9199 FJ763637 Neisseria sp. TM10_1 DQ279352 Neisseriasubflava ACEO01000067 Neorickettsia risticii CP001431 Neorickettsiasennetsu NC_007798 Nocardia brasiliensis AIHV01000038 Nocardiacyriacigeorgica HQ009486 Nocardia farcinica NC_006361 Nocardia purisNR_028994 Nocardia sp. 01_Je_025 GU574059 Nocardiopsis dassonvilleiCP002041 Novosphingobium aromaticivorans AAAV03000008 Oceanobacilluscaeni NR_041533 Oceanobacillus sp. Ndiop CAER01000083 Ochrobactrumanthropi NC_009667 Ochrobactrum intermedium ACQA01000001 Ochrobactrumpseudintermedium DQ365921 Odoribacter laneus AB490805 Odoribactersplanchnicus CP002544 Okadaella gastrococcus HQ699465 Oligellaureolytica NR_041998 Oligella urethralis NR_041753 Olsenella genomosp.C1 AY278623 Olsenella profusa FN178466 Olsenella sp. F0004 EU592964Olsenella sp. oral taxon 809 ACVE01000002 Olsenella uli CP002106Opitutus terrae NR_074978 Oribacterium sinus ACKX01000142 Oribacteriumsp. ACB1 HM120210 Oribacterium sp. ACB7 HM120211 Oribacterium sp. CM12HQ616374 Oribacterium sp. ICM51 HQ616397 Oribacterium sp. OBRC12HQ616355 Oribacterium sp. oral taxon 078 ACIQ02000009 Oribacterium sp.oral taxon 102 GQ422713 Oribacterium sp. oral taxon 108 AFIH01000001Orientia tsutsugamushi AP008981 Ornithinibacillus bavariensis NR_044923Omithinibacillus sp. 7_10AIA FN397526 Oscillibacter sp. G2 HM626173Oscillibacter valericigenes NR_074793 Oscillospira guilliermondiiAB040495 Oxalobacter formigenes ACDQ01000020 Paenibacillus barcinonensisNR_042272 Paenibacillus barengoltzii NR_042756 Paenibacillus chibensisNR_040885 Paenibacillus cookii NR_025372 Paenibacillus durus NR_037017Paenibacillus glucanolyticus D78470 Paenibacillus lactis NR_025739Paenibacillus lautus NR_040882 Paenibacillus pabuli NR_040853Paenibacillus polymyxa NR_037006 Paenibacillus popilliae NR_040888Paenibacillus sp. CIP 101062 HM212646 Paenibacillus sp. HGF5AEXS01000095 Paenibacillus sp. HGF7 AFDH01000147 Paenibacillus sp. JC66JF824808 Paenibacillus sp. oral taxon F45 HM099647 Paenibacillus sp.R_27413 HE586333 Paenibacillus sp. R_27422 HE586338 Paenibacillustimonensis NR_042844 Pantoea agglomerans AY335552 Pantoea ananatisCP001875 Pantoea brenneri EU216735 Pantoea citrea EF688008 Pantoeaconspicua EU216737 Pantoea septica EU216734 Papillibacter cinnamivoransNR_025025 Parabacteroides distasonis CP000140 Parabacteroidesgoldsteinii AY974070 Parabacteroides gordonii AB470344 Parabacteroidesjohnsonii ABYH01000014 Parabacteroides merdae EU136685 Parabacteroidessp. D13 ACPW01000017 Parabacteroides sp. NS31_3 JN029805 Parachlamydiasp. UWE25 BX908798 Paracoccus denitrificans CP000490 Paracoccus marcusiiNR_044922 Paraprevotella clara AFFY01000068 Paraprevotella xylaniphilaAFBR01000011 Parascardovia denticolens ADEB01000020 Parasutterellaexcrementihominis AFBP01000029 Parasutterella secunda AB491209Parvimonas micra AB729072 Parvimonas sp. oral taxon 110 AFII01000002Pasteurella bettyae L06088 Pasteurella dagmatis ACZR01000003 Pasteurellamultocida NC_002663 Pediococcus acidilactici ACXB01000026 Pediococcuspentosaceus NR_075052 Peptococcus niger NR_029221 Peptococcus sp. oralclone JM048 AY349389 Peptococcus sp. oral taxon I67 GQ422727Peptoniphilus asaccharolyticus D14145 Peptoniphilus duerdenii EU526290Peptoniphilus harei NR_026358 Peptoniphilus indolicus AY153431Peptoniphilus ivorii Y07840 Peptoniphilus lacrimalis ADDO01000050Peptoniphilus sp. gpac007 AM176517 Peptoniphilus sp. gpac018A AM176519Peptoniphilus sp. gpac077 AM176527 Peptoniphilus sp. gpac148 AM176535Peptoniphilus sp. JC140 JF824803 Peptoniphilus sp. oral taxon 386ADCS01000031 Peptoniphilus sp. oral taxon 836 AEAA01000090Peptostreptococcaceae bacterium ph1 JN837495 Peptostreptococcusanaerobius AY326462 Peptostreptococcus micros AM176538Peptostreptococcus sp. 9succ1 X90471 Peptostreptococcus sp. oral cloneAP24 AB175072 Peptostreptococcus sp. oral clone FJ023 AY349390Peptostreptococcus sp. P4P_31 P3 AY207059 Peptostreptococcus stomatisADGQ01000048 Phascolarctobacterium faecium NR_026111Phascolarctobacterium sp. YIT 12068 AB490812 Phascolarctobacteriumsuccinatutens AB490811 Phenylobacterium zucineum AY628697 Photorhabdusasymbiotica Z76752 Pigmentiphaga daeguensis JN585327 Planomicrobiumkoreense NR_025011 Plesiomonas shigelloides X60418 Porphyromonadaceaebacterium NML 060648 EF184292 Porphyromonas asaccharolytica AENO01000048Porphyromonas endodontalis ACNN01000021 Porphyromonas gingivalisAE015924 Porphyromonas levii NR_025907 Porphyromonas macacae NR_025908Porphyromonas somerae AB547667 Porphyromonas sp. oral clone BB134AY005068 Porphyromonas sp. oral clone F016 AY005069 Porphyromonas sp.oral clone P2PB_52 P1 AY207054 Porphyromonas sp. oral clone P4GB_100 P2AY207057 Porphyromonas sp. UQD 301 EU012301 Porphyromonas uenonisACLR01000152 Prevotella albensis NR_025300 Prevotella amnii AB547670Prevotella bergensis ACKS01000100 Prevotella bivia ADFO01000096Prevotella brevis NR_041954 Prevotella buccae ACRB01000001 Prevotellabuccalis JN867261 Prevotella copri ACBX02000014 Prevotella corporisL16465 Prevotella dentalis AB547678 Prevotella denticola CP002589Prevotella disiens AEDO01000026 Prevotella genomosp. C1 AY278624Prevotella genomosp. C2 AY278625 Prevotella genomosp. P7 oral cloneMB2_P31 DQ003620 Prevotella genomosp. P8 oral clone MB3_P13 DQ003622Prevotella genomosp. P9 oral clone MB7_G16 DQ003633 Prevotellaheparinolytica GQ422742 Prevotella histicola JN867315 Prevotellaintermedia AF414829 Prevotella loescheii JN867231 Prevotella maculosaAGEK0100003 5 Prevotella marshii AEEI01000070 Prevotella melaninogenicaCP002122 Prevotella micans AGWK01000061 Prevotella multiformisAEWX01000054 Prevotella multisaccharivorax AFJE01000016 Prevotellananceiensis JN867228 Prevotella nigrescens AFPX01000069 Prevotellaoralis AEPE01000021 Prevotella oris ADDV01000091 Prevotella oulorumL16472 Prevotella pallens AFPY01000135 Prevotella ruminicola CP002006Prevotella salivae AB108826 Prevotella sp. BI_42 AJ581354 Prevotella sp.CM38 HQ610181 Prevotella sp. ICM1 HQ616385 Prevotella sp. ICM55 HQ616399Prevotella sp. JCM 6330 AB547699 Prevotella sp. oral clone AA020AY005057 Prevotella sp. oral clone ASCG10 AY923148 Prevotella sp. oralclone ASCG12 DQ272511 Prevotella sp. oral clone AU069 AY005062Prevotella sp. oral clone CY006 AY005063 Prevotella sp. oral clone DA058AY005065 Prevotella sp. oral clone FL019 AY349392 Prevotella sp. oralclone FU048 AY349393 Prevotella sp. oral clone FW035 AY349394 Prevotellasp. oral clone GI030 AY349395 Prevotella sp. oral clone GI032 AY349396Prevotella sp. oral clone GI059 AY349397 Prevotella sp. oral clone GU027AY349398 Prevotella sp. oral clone HF050 AY349399 Prevotella sp. oralclone ID019 AY349400 Prevotella sp. oral clone IDR_CEC_0055 AY550997Prevotella sp. oral clone IK053 AY349401 Prevotella sp. oral clone IK062AY349402 Prevotella sp. oral clone P4PB_83 P2 AY207050 Prevotella sp.oral taxon 292 GQ422735 Prevotella sp. oral taxon 299 ACWZ01000026Prevotella sp. oral taxon 300 GU409549 Prevotella sp. oral taxon 302ACZK01000043 Prevotella sp. oral taxon 310 GQ422737 Prevotella sp. oraltaxon 317 ACQH01000158 Prevotella sp. oral taxon 472 ACZS01000106Prevotella sp. oral taxon 781 GQ422744 Prevotella sp. oral taxon 782GQ422745 Prevotella sp. oral taxon F68 HM099652 Prevotella sp. oraltaxon G60 GU432133 Prevotella sp. oral taxon G70 GU432179 Prevotella sp.oral taxon G71 GU432180 Prevotella sp. SEQ053 JN867222 Prevotella sp.SEQ065 JN867234 Prevotella sp. SEQ072 JN867238 Prevotella sp. SEQ116JN867246 Prevotella sp. SG12 GU561343 Prevotella sp. sp24 AB003384Prevotella sp. sp34 AB003385 Prevotella stercorea AB244774 Prevotellatannerae ACIJ02000018 Prevotella timonensis ADEF01000012 Prevotellaveroralis ACVA01000027 Prevotella jejuni, Prevotella aurantiaca,Prevotella baroniae, Prevotella colorans, Prevotella corporis,Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotellafusca, Prevotella heparinolytica, Prevotella loescheii, Prevotellamultisaccharivorax, Prevotella nanceiensis, Prevotella oryzae,Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola,Prevotella saccharolytica, Prevotella scopos, Prevotella shahii,Prevotella zoogleoformans Prevotellaceae bacterium P4P_62 P1 AY207061Prochlorococcus marinus CP000551 Propionibacteriaceae bacterium NML02_0265 EF599122 Propionibacterium acidipropionici NC_019395Propionibacterium acnes ADJM01000010 Propionibacterium avidum AJ003055Propionibacterium freudenreichii NR_036972 Propionibacterium granulosumFJ785716 Propionibacterium jensenii NR_042269 Propionibacteriumpropionicum NR_025277 Propionibacterium sp. 434_HC2 AFIL01000035Propionibacterium sp. H456 AB177643 Propionibacterium sp. LG AY354921Propionibacterium sp. oral taxon 192 GQ422728 Propionibacterium sp.S555a AB264622 Propionibacterium thoenii NR_042270 Proteus mirabilisACLE01000013 Proteus penneri ABVP01000020 Proteus sp. HS7514 DQ512963Proteus vulgaris AJ233425 Providencia alcalifaciens ABXW01000071Providencia rettgeri AM040492 Providencia rustigianii AM040489Providencia stuartii AF008581 Pseudoclavibacter sp. Timone FJ375951Pseudoflavonifractor capillosus AY136666 Pseudomonas aeruginosaAABQ07000001 Pseudomonas fluorescens AY622220 Pseudomonas gessardiiFJ943496 Pseudomonas mendocina AAUL01000021 Pseudomonas monteiliiNR_024910 Pseudomonas poae GU188951 Pseudomonas pseudoalcaligenesNR_037000 Pseudomonas putida AF094741 Pseudomonas sp. 2_1_26ACWU01000257 Pseudomonas sp. G1229 DQ910482 Pseudomonas sp. NP522bEU723211 Pseudomonas stutzeri AM905854 Pseudomonas tolaasii AF320988Pseudomonas viridiflava NR_042764 Pseudoramibacter alactolyticusAB036759 Psychrobacter arcticus CP000082 Psychrobacter cibarius HQ698586Psychrobacter cryohalolentis CP000323 Psychrobacter faecalis HQ698566Psychrobacter nivimaris HQ698587 Psychrobacter pulmonis HQ698582Psychrobacter sp. 13983 HM212668 Pyramidobacter piscolens AY207056Ralstonia pickettii NC_010682 Ralstonia sp. 5_7_47FAA ACUF01000076Raoultella omithinolytica AB364958 Raoultella planticola AF129443Raoultella terrigena NR_037085 Rhodobacter sp. oral taxon C30 HM099648Rhodobacter sphaeroides CP000144 Rhodococcus corynebacterioides X80615Rhodococcus equi ADNW01000058 Rhodococcus erythropolis ACNO01000030Rhodococcus fascians NR_037021 Rhodopseudomonas palustris CP000301Rickettsia akari CP000847 Rickettsia conorii AE008647 Rickettsiaprowazekii M21789 Rickettsia rickettsii NC_010263 Rickettsia slovacaL36224 Rickettsia typhi AE017197 Robinsoniella peoriensis AF445258Roseburia cecicola GU233441 Roseburia faecalis AY804149 Roseburia faecisAY305310 Roseburia hominis AJ270482 Roseburia intestinalis FP929050Roseburia inulinivorans AJ270473 Roseburia sp. 11SE37 FM954975 Roseburiasp. 11SE38 FM954976 Roseiflexus castenholzii CP000804 Roseomonascervicalis ADVL01000363 Roseomonas mucosa NR_028857 Roseomonas sp.NML94_0193 AF533357 Roseomonas sp. NML97_0121 AF533359 Roseomonas sp.NML98_0009 AF533358 Roseomonas sp. NML98_0157 AF533360 Rothia aeriaDQ673320 Rothia dentocariosa ADDW01000024 Rothia mucilaginosaACVO01000020 Rothia nasimurium NR_025310 Rothia sp. oral taxon 188GU470892 Ruminobacter amylophilus NR_026450 Ruminococcaceae bacteriumD16 ADDX01000083 Ruminococcus albus AY445600 Ruminococcus bromiiEU266549 Ruminococcus callidus NR_029160 Ruminococcus champanellensisFP929052 Ruminococcus flavefaciens NR_025931 Ruminococcus gnavus X94967Ruminococcus hansenii M59114 Ruminococcus lactaris ABOU02000049Ruminococcus obeum AY169419 Ruminococcus sp. 18P13 AJ515913 Ruminococcussp. 5_1_39BFAA ACII01000172 Ruminococcus sp. 9SE51 FM954974 Ruminococcussp. ID8 AY960564 Ruminococcus sp. K_1 AB222208 Ruminococcus torquesAAVP02000002 Saccharomonospora viridis X54286 Salmonella bongoriNR_041699 Salmonella enterica NC_011149 Salmonella enterica NC_011205Salmonella enterica DQ344532 Salmonella enterica ABEH02000004 Salmonellaenterica ABAK02000001 Salmonella enterica NC_011080 Salmonella entericaEU118094 Salmonella enterica NC_011094 Salmonella enterica AE014613Salmonella enterica ABFH02000001 Salmonella enterica ABEM01000001Salmonella enterica ABAM02000001 Salmonella typhimurium DQ344533Salmonella typhimurium AF170176 Sarcina ventriculi NR_026146 Scardoviainopinata AB029087 Scardovia wiggsiae AY278626 Segniliparus rotundusCP001958 Segniliparus rugosus ACZI01000025 Selenomonas artemidisHM596274 Selenomonas dianae GQ422719 Selenomonas flueggei AF287803Selenomonas genomosp. C1 AY278627 Selenomonas genomosp. C2 AY278628Selenomonas genomosp. P5 AY341820 Selenomonas genomosp. P6 oral cloneMB3_C41 DQ003636 Selenomonas genomosp. P7 oral clone MB5_C08 DQ003627Selenomonas genomosp. P8 oral clone MB5_P06 DQ003628 Selenomonas infelixAF287802 Selenomonas noxia GU470909 Selenomonas ruminantium NR_075026Selenomonas sp. FOBRC9 HQ616378 Selenomonas sp. oral clone FT050AY349403 Selenomonas sp. oral clone GI064 AY349404 Selenomonas sp. oralclone GT010 AY349405 Selenomonas sp. oral clone HU051 AY349406Selenomonas sp. oral clone IK004 AY349407 Selenomonas sp. oral cloneIQ048 AY349408 Selenomonas sp. oral clone JI021 AY349409 Selenomonas sp.oral clone JS031 AY349410 Selenomonas sp. oral clone OH4A AY947498Selenomonas sp. oral clone P2PA_80 P4 AY207052 Selenomonas sp. oraltaxon 137 AENV01000007 Selenomonas sp. oral taxon 149 AEEJ01000007Selenomonas sputigena ACKP02000033 Serratia fonticola NR_025339 Serratialiquefaciens NR_042062 Serratia marcescens GU826157 Serratia odoriferaADBY01000001 Serratia proteamaculans AAUN01000015 Shewanellaputrefaciens CP002457 Shigella boydii AAKA01000007 Shigella dysenteriaeNC_007606 Shigella flexneri AE005674 Shigella sonnei NC_007384Shuttleworthia satelles ACIP02000004 Shuttleworthia sp. MSX8B HQ616383Shuttleworthia sp. oral taxon G69 GU432167 Simonsiella muelleriADCY01000105 Slackia equolifaciens EU3 77663 Slackia exigua ACUX01000029Slackia faecicanis NR_042220 Slackia heliotrinireducens NR_074439Slackia isoflavoniconvertens AB566418 Slackia piriformis AB490806Slackia sp. NATTS AB505075 Solobacterium moorei AECQ01000039Sphingobacterium faecium NR_025537 Sphingobacterium mizutaii JF708889Sphingobacterium multivorum NR_040953 Sphingobacterium spiritivorumACHA02000013 Sphingomonas echinoides NR_024700 Sphingomonas sp. oralclone FI012 AY349411 Sphingomonas sp. oral clone FZ016 AY349412Sphingomonas sp. oral taxon A09 HM099639 Sphingomonas sp. oral taxon F71HM099645 Sphingopyxis alaskensis CP000356 Spiroplasma insolitumNR_025705 Sporobacter termitidis NR_044972 Sporolactobacillus inulinusNR_040962 Sporolactobacillus nakayamae NR_042247 Sporosarcinanewyorkensis AFPZ01000142 Sporosarcina sp. 2681 GU994081Staphylococcaceae bacterium NML 92_0017 AY841362 Staphylococcus aureusCP002643 Staphylococcus auricularis JQ624774 Staphylococcus capitisACFR01000029 Staphylococcus caprae ACRH01000033 Staphylococcus camosusNR_075003 Staphylococcus cohnii JN175375 Staphylococcus condimentiNR_029345 Staphylococcus epidermidis ACHE01000056 Staphylococcus equorumNR_027520 Staphylococcus fleurettii NR_041326 Staphylococcushaemolyticus NC_007168 Staphylococcus hominis AM157418 Staphylococcuslugdunensis AEQA01000024 Staphylococcus pasteuri FJ189773 Staphylococcuspseudintermedius CP002439 Staphylococcus saccharolyticus NR_029158Staphylococcus saprophyticus NC_007350 Staphylococcus sciuri NR_025520Staphylococcus sp. clone bottae7 AF467424 Staphylococcus sp. H292AB177642 Staphylococcus sp. H780 AB177644 Staphylococcus succinusNR_028667 Staphylococcus vitulinus NR_024670 Staphylococcus wameriACPZ01000009 Staphylococcus xylosus AY395016 Stenotrophomonasmaltophilia AAVZ01000005 Stenotrophomonas sp. FG_6 EF017810Streptobacillus moniliformis NR_027615 Streptococcus agalactiaeAAJ001000130 Streptococcus alactolyticus NR_041781 Streptococcusanginosus AECT01000011 Streptococcus australis AEQR01000024Streptococcus bovis AEEL01000030 Streptococcus canis AJ413203Streptococcus constellatus AY277942 Streptococcus cristatus AEVC01000028Streptococcus downei AEKN01000002 Streptococcus dysgalactiae AP010935Streptococcus equi CP001129 Streptococcus equinus AEVB01000043Streptococcus gallolyticus FR824043 Streptococcus genomosp. C1 AY278629Streptococcus genomosp. C2 AY278630 Streptococcus genomosp. C3 AY278631Streptococcus genomosp. C4 AY278632 Streptococcus genomosp. C5 AY278633Streptococcus genomosp. C6 AY278634 Streptococcus genomosp. C7 AY278635Streptococcus genomosp. C8 AY278609 Streptococcus gordonii NC_009785Streptococcus infantarius ABJK02000017 Streptococcus infantisAFNN01000024 Streptococcus intermedius NR_028736 Streptococcuslutetiensis NR_037096 Streptococcus massiliensis AY769997 Streptococcusmilleri X81023 Streptococcus mitis AM157420 Streptococcus mutansAP010655 Streptococcus oligofermentans AY099095 Streptococcus oralisADMV01000001 Streptococcus parasanguinis AEKM01000012 Streptococcuspasteurianus AP012054 Streptococcus peroris AEVF01000016 Streptococcuspneumoniae AE008537 Streptococcus porcinus EF121439 Streptococcuspseudopneumoniae FJ827123 Streptococcus pseudoporcinus AENS01000003Streptococcus pyogenes AE006496 Streptococcus ratti X58304 Streptococcussalivarius AGBV01000001 Streptococcus sanguinis NR_074974 Streptococcussinensis AF432857 Streptococcus sp. 16362 JN590019 Streptococcus sp.2_1_36FAA ACOI01000028 Streptococcus sp. 2285_97 AJ131965 Streptococcussp. 69130 X78825 Streptococcus sp. AC15 HQ616356 Streptococcus sp. ACS2HQ616360 Streptococcus sp. AS20 HQ616366 Streptococcus sp. BS35aHQ616369 Streptococcus sp. C150 ACRI01000045 Streptococcus sp. CM6HQ616372 Streptococcus sp. CM7 HQ616373 Streptococcus sp. ICM10 HQ616389Streptococcus sp. ICM12 HQ616390 Streptococcus sp. ICM2 HQ616386Streptococcus sp. ICM4 HQ616387 Streptococcus sp. ICM45 HQ616394Streptococcus sp. M143 ACRK01000025 Streptococcus sp. M334 ACRL01000052Streptococcus sp. OBRC6 HQ616352 Streptococcus sp. oral clone ASB02AY923121 Streptococcus sp. oral clone ASCA03 DQ272504 Streptococcus sp.oral clone ASCA04 AY923116 Streptococcus sp. oral clone ASCA09 AY923119Streptococcus sp. oral clone ASCB04 AY923123 Streptococcus sp. oralclone ASCB06 AY923124 Streptococcus sp. oral clone ASCC04 AY923127Streptococcus sp. oral clone ASCC05 AY923128 Streptococcus sp. oralclone ASCC12 DQ272507 Streptococcus sp. oral clone ASCD01 AY923129Streptococcus sp. oral clone ASCD09 AY923130 Streptococcus sp. oralclone ASCD10 DQ272509 Streptococcus sp. oral clone ASCE03 AY923134Streptococcus sp. oral clone ASCE04 AY953253 Streptococcus sp. oralclone ASCE05 DQ272510 Streptococcus sp. oral clone ASCE06 AY923135Streptococcus sp. oral clone ASCE09 AY923136 Streptococcus sp. oralclone ASCE10 AY923137 Streptococcus sp. oral clone ASCE12 AY923138Streptococcus sp. oral clone ASCF05 AY923140 Streptococcus sp. oralclone ASCF07 AY953255 Streptococcus sp. oral clone ASCF09 AY923142Streptococcus sp. oral clone ASCG04 AY923145 Streptococcus sp. oralclone BW009 AY005042 Streptococcus sp. oral clone CH016 AY005044Streptococcus sp. oral clone GK051 AY349413 Streptococcus sp. oral cloneGM006 AY349414 Streptococcus sp. oral clone P2PA_41 P2 AY207051Streptococcus sp. oral clone P4PA_30 P4 AY207064 Streptococcus sp. oraltaxon 071 AEEP01000019 Streptococcus sp. oral taxon G59 GU432132Streptococcus sp. oral taxon G62 GU432146 Streptococcus sp. oral taxonG63 GU432150 Streptococcus sp. SHV515 Y07601 Streptococcus suis FM252032Streptococcus thermophilus CP000419 Streptococcus uberis HQ391900Streptococcus urinalis DQ303194 Streptococcus vestibularis AEKO01000008Streptococcus viridans AF076036 Streptomyces albus AJ697941 Streptomycesgriseus NR_074787 Streptomyces sp. 1 AIP_2009 FJ176782 Streptomyces sp.SD 511 EU544231 Streptomyces sp. SD 524 EU544234 Streptomyces sp. SD 528EU544233 Streptomyces sp. SD 534 EU544232 Streptomyces thermoviolaceusNR_027616 Subdoligranulum variabile AJ518869 Succinatimonas hippeiAEVO01000027 Sutterella morbirenis AJ832129 Sutterella parvirubraAB300989 Sutterella sanguinus AJ748647 Sutterella sp. YIT 12072 AB491210Sutterella stercoricanis NR_025600 Sutterella wadsworthensisADMF01000048 Synergistes genomosp. C1 AY278615 Synergistes sp. RMA 14551DQ412722 Synergistetes bacterium ADV897 GQ258968 Synergistetes bacteriumLBVCM1157 GQ258969 Synergistetes bacterium oral taxon 362 GU410752Synergistetes bacterium oral taxon D48 GU430992 Syntrophococcussucromutans NR_036869 Syntrophomonadaceae genomosp. P1 AY341821Tannerella forsythia CP003191 Tannerella sp. 6_1_58FAA_CT1 ACWX01000068Tatlockia micdadei M36032 Tatumella ptyseos NR_025342 Tessaracoccus sp.oral taxon F04 HM099640 Tetragenococcus halophilus NR_075020Tetragenococcus koreensis NR_043113 Thermoanaerobacter pseudethanolicusCP000924 Thermobifida fusca NC_007333 Thermofilum pendens X14835 Thermusaquaticus NR_025900 Tissierella praeacuta NR_044860 Trabulsiellaguamensis AY373830 Treponema denticola ADEC01000002 Treponema genomosp.P1 AY341822 Treponema genomosp. P4 oral clone MB2_G19 DQ003618 Treponemagenomosp. P5 oral clone MB3_P23 DQ003624 Treponema genomosp. P6 oralclone MB4_G11 DQ003625 Treponema lecithinolyticum NR_026247 Treponemapallidum CP001752 Treponema parvum AF302937 Treponema phagedenisAEFH01000172 Treponema putidum AJ543428 Treponema refringens AF426101Treponema socranskii NR_024868 Treponema sp. 6:H:D15A_4 AY005083Treponema sp. clone DDKL_4 Y08894 Treponema sp. oral clone JU025AY349417 Treponema sp. oral clone JU031 AY349416 Treponema sp. oralclone P2PB_53 P3 AY207055 Treponema sp. oral taxon 228 GU408580Treponema sp. oral taxon 230 GU408603 Treponema sp. oral taxon 231GU408631 Treponema sp. oral taxon 232 GU408646 Treponema sp. oral taxon235 GU408673 Treponema sp. oral taxon 239 GU408738 Treponema sp. oraltaxon 247 GU408748 Treponema sp. oral taxon 250 GU408776 Treponema sp.oral taxon 251 GU408781 Treponema sp. oral taxon 254 GU408803 Treponemasp. oral taxon 265 GU408850 Treponema sp. oral taxon 270 GQ422733Treponema sp. oral taxon 271 GU408871 Treponema sp. oral taxon 508GU413616 Treponema sp. oral taxon 518 GU413640 Treponema sp. oral taxonG85 GU432215 Treponema sp. ovine footrot AJO10951 Treponema vincentiiACYH0100003 6 Tropheryma whipplei BX251412 Trueperella pyogenesNR_044858 Tsukamurella paurometabola X80628 Tsukamurella tyrosinosolvensAB478958 Turicibacter sanguinis AF349724 Ureaplasma parvum AE002127Ureaplasma urealyticum AAYN01000002 Ureibacillus composti NR_043746Ureibacillus suwonensis NR_043232 Ureibacillus terrenus NR_025394Ureibacillus thermophilus NR_043747 Ureibacillus thermosphaericusNR_040961 Vagococcus fluvialis NR_026489 Veillonella atypicaAEDS01000059 Veillonella dispar ACIK02000021 Veillonella genomosp. P1oral clone MB5_P17 DQ003631 Veillonella montpellierensis AF473836Veillonella parvula ADFU01000009 Veillonella sp. 3_1_44 ADCV01000019Veillonella sp. 6_1_27 ADCW01000016 Veillonella sp. ACP1 HQ616359Veillonella sp. AS16 HQ616365 Veillonella sp. BS32b HQ616368 Veillonellasp. ICM51a HQ616396 Veillonella sp. MSA12 HQ616381 Veillonella sp. NVG100cf EF108443 Veillonella sp. OK11 JN695650 Veillonella sp. oral cloneASCA08 AY923118 Veillonella sp. oral clone ASCB03 AY923122 Veillonellasp. oral clone ASCG01 AY923144 Veillonella sp. oral clone ASCG02AY953257 Veillonella sp. oral clone OH1A AY947495 Veillonella sp. oraltaxon 158 AENU01000007 Veillonellaceae bacterium oral taxon 131 GU402916Veillonellaceae bacterium oral taxon 155 GU470897 Vibrio choleraeAAUR01000095 Vibrio fluvialis X76335 Vibrio furnissii CP002377 Vibriomimicus ADAF01000001 Vibrio parahaemolyticus AAWQ01000116 Vibrio sp.RC341 ACZT01000024 Vibrio vulnificus AE016796 Victivallaceae bacteriumNML 080035 FJ394915 Victivallis vadensis ABDE02000010 Virgibacillusproomii NR_025308 Weissella beninensis EU439435 Weissella cibariaNR_036924 Weissella confusa NR_040816 Weissella hellenica AB680902Weissella kandleri NR_044659 Weissella koreensis NR_075058 Weissellaparamesenteroides ACKU01000017 Weissella sp. KLDS 7.0701 EU600924Wolinella succinogenes BX571657 Xanthomonadaceae bacterium NML 03_0222EU313791 Xanthomonas campestris EF101975 Xanthomonas sp. kmd_489EU723184 Xenophilus aerolatus JN585329 Yersinia aldovae AJ871363Yersinia aleksiciae AJ627597 Yersinia bercovieri AF366377 Yersiniaenterocolitica FR729477 Yersinia frederiksenii AF366379 Yersiniaintermedia AF366380 Yersinia kristensenii ACCA01000078 Yersiniamollaretii NR_027546 Yersinia pestis AE013632 Yersiniapseudotuberculosis NC_009708 Yersinia rohdei ACCD01000071 Yokenellaregensburgei AB273739 Zimmermannella bifida AB012592 Zymomonas mobilisNR_074274

TABLE 2 Exemplary Oncophilic Bacteria Genera Species Tumor AssociationMycoplasma hyorhinis Gastric Carcinoma Propionibacterium Acnes ProstateCancer Mycoplasma genitalium Prostate Cancer Methylophilus sp. ProstateCancer Chlamydia trachomatis Prostate Cancer Helicobacter pylori GastricMALT Listeria welshimeri Renal Cancer Streptococcus pneumoniae Lymphomaand Leukemia Haemophilus influenzae Lymphoma and Leukemia Staphylococcusaureus Breast Cancer Listeria monocytogenes Breast CancerMethylobacterium radiotolerans Breast Cancer Shingomonas yanoikuyaebreast Cancer Fusobacterium sp Larynx cancer Provetelis sp Larynx cancerstreptococcus pneumoniae Larynx cancer Gemella sp Larynx cancerBordetella Pertussis Larynx cancer Corumebacterium tuberculosteraricumOral squamous cell carcinoma Micrococcus luteus Oral squamous cellcarcinoma Prevotella melaninogenica Oral squamous cell carcinomaExiguobacterium oxidotolerans Oral squamous cell carcinoma Fusobacteriumnaviforme Oral squamous cell carcinoma Veillonella parvula Oral squamouscell carcinoma Streptococcus salivarius Oral squamous cell carcinomaStreptococcus mitis/oralis Oral squamous cell carcinoma veillonelladispar Oral squamous cell carcinoma Peptostreptococcus stomatis Oralsquamous cell carcinoma Streptococcus gordonii Oral squamous cellcarcinoma Gemella Haemolysans Oral squamous cell carcinoma Gemellamorbillorum Oral squamous cell carcinoma Johnsonella ignava Oralsquamous cell carcinoma Streptococcus parasanguins Oral squamous cellcarcinoma Granulicatella adiacens Oral squamous cell carcinomaMycobacteria marinum lung infection Campylobacter concisus Barrett'sEsophagus Campylobacter rectus Barrett's Esophagus Oribacterium spEsophageal adenocarcinoma Catonella sp Esophageal adenocarcinomaPeptostreptococcus sp Esophageal adenocarcinoma Eubacterium spEsophageal adenocarcinoma Dialister sp Esophageal adenocarcinomaVeillonella sp Esophageal adenocarcinoma Anaeroglobus sp Esophagealadenocarcinoma Megasphaera sp Esophageal adenocarcinoma Atoppbium spEsophageal adenocarcinoma Solobacterium sp Esophageal adenocarcinomaRothia sp Esophageal adenocarcinoma Actinomyces sp Esophagealadenocarcinoma Fusobacterium sp Esophageal adenocarcinoma Sneathia spEsophageal adenocarcinoma Leptotrichia sp Esophageal adenocarcinomaCapnocytophaga sp Esophageal adenocarcinoma Prevotella sp Esophagealadenocarcinoma Porphyromonas sp Esophageal adenocarcinoma Campylobactersp Esophageal adenocarcinoma Haemophilus sp Esophageal adenocarcinomaNeisseria sp Esophageal adenocarcinoma TM7 sp Esophageal adenocarcinomaGranulicatella sp Esophageal adenocarcinoma Variovorax sp PsuedomyxomaPeritonei Escherichia Shigella Psuedomyxoma Peritonei Pseudomonas spPsuedomyxoma Peritonei Tessaracoccus sp Psuedomyxoma PeritoneiAcinetobacter sp Psuedomyxoma Peritonei Helicobacter hepaticus Breastcancer Chlamydia psittaci MALT lymphoma Borrelia burgdorferi B celllymphoma skin Escherichia Coli NC101 Colorectal Cancer Salmonellatyphimurium Tool Eterococcus faecalis blood Streptococcus mitis bloodStreptococcus sanguis blood Streptococcus anginosus blood Streptococcussalvarius blood Staphylococcus epidermidis blood Streptococcusgallolyticus Colorectal Cancer Campylobacter showae CC57C ColorectalCancer Leptotrichia sp Colorectal Cancer

In certain embodiments the EVs described herein areobtained fromobligate anaerobic bacteria. Examples of obligate anaerobic bacteriainclude gram-negative rods (including the genera of Bacteroides,Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterellaspp.), gram-positive cocci (primarily Peptostreptococcus spp.),gram-positive spore-forming (Clostraidium spp.), non-spore-formingbacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus andBobacterium spp.), and gram-negative cocci (mainly Veillonella spp.). Insome embodiments, the obligate anearoic bacteria are of a genus selectedfrom Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma,Longicatena, Paraclostridium, Turicibacter, and Tyzzerella.

In some embodiments, the EVs described herein are obtained frombacterium of a genus selected from Escherichia, Klebsiella,Lactobacillus, Shigella, and Staphylococcusa.

In some embodiments, the bacteria and/or EVs described herein are of aspecies selected from Blautia massiliensis, Paraclostridiumbenzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Veillonellatobetsuensis.

In some embodiments, the EVs and/or bacteria described herein aremodified such that they comprise, are linked to, and/or are bound by atherapeutic moiety. In some embodiments, the therapeutic moiety is acancer-specific moiety. In some embodiments, the cancer-specific moietyhas binding specificity for a cancer cell (e.g., has binding specificityfor a cancer-specific antigen). In some embodiments, the cancer-specificmoiety comprises an antibody or antigen binding fragment thereof. Insome embodiments, the cancer-specific moiety comprises a T cell receptoror a chimeric antigen receptor (CAR). In some embodiments, thecancer-specific moiety comprises a ligand for a receptor expressed onthe surface of a cancer cell or a receptor-binding fragment thereof. Insome embodiments, the cancer-specific moiety is a bipartite fusionprotein that has two parts: a first part that binds to and/or is linkedto the bacterium and a second part that is capable of binding to acancer cell (e.g., by having binding specificity for a cancer-specificantigen). In some embodiments, the first part is a fragment of or afull-length peptidoglycan recognition protein, such as PGRP. In someembodiments the first part has binding specificity for the EV (e.g., byhaving binding specificity for a bacterial antigen). In someembodiments, the first and/or second part comprises an antibody orantigen binding fragment thereof. In some embodiments, the first and/orsecond part comprises a T cell receptor or a chimeric antigen receptor(CAR). In some embodiments, the first and/or second part comprises aligand for a receptor expressed on the surface of a cancer cell or areceptor-binding fragment thereof. In certain embodiments,co-administration of the cancer-specific moiety with the EVs (either incombination or in separate administrations) increases the targeting ofthe EVs to the cancer cells. [87] In some embodiments, the EVs describedherein is modified such that they comprise, are linked to, and/or arebound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead).In some embodiments, the magnetic and/or paramagnetic moiety iscomprised by and/or directly linked to the bacteria. In someembodiments, the magnetic and/or paramagnetic moiety is linked to and/ora part of an EV-binding moiety that that binds to the EV. In someembodiments, the EV-binding moiety is a fragment of or a full-lengthpeptidoglycan recognition protein, such as PGRP. In some embodiments theEV-binding moiety has binding specificity for the EV(e.g., by havingbinding specificity for a bacterial antigen). In some embodiments, theEV-binding moiety comprises an antibody or antigen binding fragmentthereof. In some embodiments, the EV-binding moiety comprises a T cellreceptor or a chimeric antigen receptor (CAR). In some embodiments, theEV-binding moiety comprises a ligand for a receptor expressed on thesurface of a cancer cell or a receptor-binding fragment thereof. Incertain embodiments, co-administration of the magnetic and/orparamagnetic moiety with the EVs (either together or in separateadministrations) can be used to increase the targeting of the EVs tocancer calls and/or a part of a subject where cancer cells are present.

Production of EVs

In certain aspects, the EVs described herein can be prepared using anymethod known in the art.

In some embodiments, the EVs are prepared without an EV purificationstep. For example, in some embodiments, bacteria comprising the EVsdescribed herein are killed using a method that leaves the bacterial EVsintact and the resulting bacterial components, including the EVs, areused in the methods and compositions described herein. In someembodiments, the bacteria are killed using an antibiotic (e.g., using anantibiotic described herein). In some embodiments, the bacteria arekilled using UV irradiation.

In some embodiments, the EVs described herein are purified from one ormore other bacterial components. Methods for purifying EVs from bacteriaare known in the art. In some embodiments EVs are prepared frombacterial cultures using methods described in S. Bin Park, et al. PLoSONE. 6(3):e17629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353(2015), each of which is hereby incorporated by reference in itsentirety. In some embodiments, the bacteria are cultured to high opticaldensity and then centrifuged to pellet bacteria (e.g., at 10,000 ×g for30 min at 4° C.). In some embodiments, the culture supernatants are thenpassed through filter to exclude intact bacterial cells (e.g., a 0.22 μmfilter). In some embodiments, filtered supernatants are centrifuged topellet bacterial EVs (e.g., at 100,000-150,000×g for 1-3 hours at 4°C.). In some embodiments, the EVs are further purified by resuspendingthe resulting EV pellets (e.g., in PBS), and applying the resuspendedEVs to sucrose gradient (e.g., a 30-60% discontinuous sucrose gradient),followed by centrifugation (e.g., at 200,000×g for 20 hours at 4° C.).EV bands can be collected, washed with (e.g., with PBS), and centrifugedto pellet the EVs (e.g., at 150,000×g for 3 hours at 4° C.). Thepurified EVs can be stored, for example, at −80° C. until use. In someembodiments, the EVs are further purified by treatment with DNase and/orproteinase K.

For example, in some embodiments, cultures of bacteria disclosed hereincan be centrifuged at 11,000×g for 20-40 min at 4° C. to pelletbacteria. Culture supernatants may be passed through a 0.22 μm filter toexclude intact bacterial cells. Filtered supernatants may then beconcentrated using methods that may include, but are not limited to,ammonium sulfate precipitation, ultracentrifugation, or filtration. Forexample, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfatecan be added to filtered supernatant slowly, while stirring at 4° C.Precipitations can be incubated at 4° C. for 8-48 hours and thencentrifuged at 11,000×g for 20-40 min at 4° C. The resulting pelletscontain bacterial EVs and other debris. Using ultracentrifugation,filtered supernatants can be centrifuged at 100,000-200,000×g for 1-16hours at 4° C. The pellet of this centrifugation contains bacterial EVsand other debris. In some embodiments, using a filtration technique,such as through the use of an Amicon Ultra spin filter or by tangentialflow filtration, supernatants can be filtered so as to retain species ofmolecular weight >50 or 100 kDa. [92] Alternatively, EVs can be obtainedfrom bacterial cultures continuously during growth, or at selected timepoints during growth, by connecting a bioreactor to an alternatingtangential flow (ATF) system (e.g., XCell ATF from Repligen). The ATFsystem retains intact cells (>0.22 um) in the bioreactor, and allowssmaller components (e.g., EVs, free proteins) to pass through a filterfor collection. For example, the system may be configured so that the<0.22 um filtrate is then passed through a second filter of 100 kDa,allowing species such as EVs between 0.22 um and 100 kDa to becollected, and species smaller than 100 kDa to be pumped back into thebioreactor. Alternatively, the system may be configured to allow formedium in the bioreactor to be replenished and/or modified during growthof the culture. EVs collected by this method may be further purifiedand/or concentrated by ultracentrifugation or filtration as describedabove for filtered supernatants.

EVs obtained by methods provided herein may be further purified by sizebased column chromatography, by affinity chromatography, and by gradientultracentrifugation, using methods that may include, but are not limitedto, use of a sucrose gradient or Optiprep gradient. Briefly, using asucrose gradient method, if ammonium sulfate precipitation orultracentrifugation were used to concentrate the filtered supernatants,pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. Iffiltration was used to concentrate the filtered supernatant, theconcentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0,using an Amicon Ultra column. Samples are applied to a 35-60%discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24hours at 4° C. Briefly, using an Optiprep gradient method, if ammoniumsulfate precipitation or ultracentrifugation were used to concentratethe filtered supernatants, pellets are resuspended in 35% Optiprep inPBS. In some embodiments, if filtration was used to concentrate thefiltered supernatant, the concentrate is diluted using 60% Optiprep to afinal concentration of 35% Optiprep. Samples are applied to a 35-60%discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24hours at 4° C.

In some embodiments, to confirm sterility and isolation of the EVpreparations, EVs are serially diluted onto agar medium used for routineculture of the bacteria being tested, and incubated using routineconditions. Non-sterile preparations are passed through a 0.22 um filterto exclude intact cells. To further increase purity, isolated EVs may beDNase or proteinase K treated.

In some embodiments, for preparation of EVs used for in vivo injections,purified EVs are processed as described previously (G. Norheim, et al.PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradientcentrifugation, bands containing EVs are resuspended to a finalconcentration of 50 μg/mL in a solution containing 3% sucrose or othersolution suitable for in vivo injection known to one skilled in the art.This solution may also contain adjuvant, for example aluminum hydroxideat a concentration of 0-0.5% (w/v).

In certain embodiments, to make samples compatible with further testing(e.g. to remove sucrose prior to TEM imaging or in vitro assays),samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 usingfiltration (e.g. Amicon Ultra columns), dialysis, or ultracentrifugation(200,000×g, ≥3 hours, 4° C.) and resuspension.

In some embodiments, the sterility of the EV preparations can beconfirmed by plating a portion of the EVs onto agar medium used forstandard culture of the bacteria used in the generation of the EVs andincubating using standard conditions.

In some embodiments select EVs are isolated and enriched bychromatography and binding surface moieties on EVs. In otherembodiments, select EVs are isolated and/or enriched by fluorescent cellsorting by methods using affinity reagents, chemical dyes, recombinantproteins or other methods known to one skilled in the art.

Pharmaceutical Compositions

In certain embodiments, the methods provided herein are pharmaceuticalcompositions comprising EVs and/or bacteria provided herein (e.g., an EVcomposition). In some embodiments, the EV composition comprises an EVand/or a combination of EVs described herein and a pharmaceuticallyacceptable carrier.

In some embodiments, the pharmaceutical compositions comprise EVssubstantially or entirely free of bacteria. In some embodiments, thepharmaceutical compositions comprise both EVs and whole bacteria (e.g.,live bacteria, killed bacteria, attenuated bacteria). In certainembodiments, the pharmaceutical compositions comprise bacteria that issubstantially or entirely free of EVs. In some embodiments, thepharmaceutical compositions comprise EVs and/or bacteria from one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the bacteriastrains or species listed in Table 1 and/or Table 2.

In some embodiments, the pharmaceutical composition comprises at least 1bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10¹² EV particles.

In some embodiments, the pharmaceutical composition comprises about 1bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10¹² EV particles.

In certain embodiments, the pharmaceutical composition comprises acertain ratio of bacteria particles to EV particles. The number ofbacteria particles can be based on actual particle number or (if thebacteria is live) the number of CFUs. The particle number can beestablished by combining a set number of purified EVs with a set numberof purified bacterium, by modifying the growth conditions under whichthe bacteria are cultured, or by modifying the bacteria itself toproduce more or fewer EVs.

In some embodiments, to quantify the numbers of EVs and/or bacteriapresent in a bacterial sample, electron microscopy (e.g., EM ofultrathin frozen sections) can be used to visualize the vesicles andbacteria and count their relative numbers. Alternatively, combinationsof nanoparticle tracking analysis (NTA), Coulter counting, and dynamiclight scattering (DLS) or a combination of these techniques can be used.NTA and the Coulter counter count particles and show their sizes. DLSgives the size distribution of particles, but not the concentration.Bacteria frequently have diameters of 1-2 um. The full range is 0.2-20um. Combined results from Coulter counting and NTA can reveal thenumbers of bacteria in a given sample. Coulter counting reveals thenumbers of particles with diameters of 0.7-10 um. NTA reveals thenumbers of particles with diameters of 50-1400 nm. For most bacterialsamples, the Coulter counter alone can reveal the number of bacteria ina sample. EVs are 20-250 nm in diameter. NTA will allow us to count thenumbers of particles that are 50-250 nm in diameter. DLS reveals thedistribution of particles of different diameters within an approximaterange of 1 nm - 3 um.

In some embodiments, the pharmaceutical composition comprises no morethan 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³,4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴,5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵,6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷,8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸,9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹,1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰,1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹,and/or 1×10¹² EV particles.

In some embodiments, the pharmaceutical composition comprises at least 1EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷ , 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10 ¹² bacterium.

In some embodiments, the pharmaceutical composition comprises about 1 EVparticle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷ , 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁹, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10′² bacterium. In some embodiments, the pharmaceutical compositioncomprises no more than 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³,2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴,3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵,4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰,8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹,8×10¹¹, 9×10¹¹, and/or 1×10 ¹² bacterium.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are EVs.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are bacteria.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are EVs.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are bacteria.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the particles in the pharmaceutical compositionare EVs.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the particles in the pharmaceutical compositionare bacteria.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is EV protein.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is bacteria protein.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is EV protein.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is bacteria protein.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the protein in the pharmaceutical composition isEV protein.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the protein in the pharmaceutical composition isbacteria protein.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are EV lipids.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are bacteria lipids.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are EV lipids.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are bacteria lipids.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%,

44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%of the lipids in the pharmaceutical composition are EV lipids.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition arebacteria lipids.

In some embodiments, the EVs in the pharmaceutical composition arepurified from one or more other bacterial components. In someembodiments, the pharmaceutical composition further comprises otherbacterial components. In some embodiments, the pharmaceuticalcomposition comprise bacteria cells.

In certain aspects, provided are pharmaceutical compositions foradministration subjects. In some embodiments, the pharmaceuticalcompositions are combined with additional active and/or inactivematerials in order to produce a final product, which may be in singledosage unit or in a multi-dose format. In some embodiments, the thepharmaceutical compositions is combined with an adjuvant such as animmuno-adjuvant (e.g., STING agonists, TLR agonists, NOD agonists).

In some embodiments the composition comprises at least one carbohydrate.A “carbohydrate” refers to a sugar or polymer of sugars. The terms“saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide”may be used interchangeably. Most carbohydrates are aldehydes or ketoneswith many hydroxyl groups, usually one on each carbon atom of themolecule. Carbohydrates generally have the molecular formulaC_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replaced with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments the composition comprises at least one lipid. Asused herein a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In some embodiments thecomposition comprises at least one modified lipid, for example a lipidthat has been modified by cooking.

In some embodiments the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments the composition comprises at least one supplementalvitamin. The at least one vitamin can be fat-soluble or water solublevitamins. Suitable vitamins include but are not limited to vitamin C,vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin,vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenicacid, and biotin. Suitable forms of any of the foregoing are salts ofthe vitamin, derivatives of the vitamin, compounds having the same orsimilar activity of the vitamin, and metabolites of the vitamin.

In some embodiments the composition comprises an excipient. Non-limitingexamples of suitable excipients include a buffering agent, apreservative, a stabilizer, a binder, a compaction agent, a lubricant, adispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In some embodiments the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments the excipient comprises a preservative. Non-limitingexamples of suitable preservatives include antioxidants, such asalpha-tocopherol and ascorbate, and antimicrobials, such as parabens,chlorobutanol, and phenol.

In some embodiments the composition comprises a binder as an excipient.Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In some embodiments the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In some embodiments the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments the composition comprises a disintegrant as anexcipient. In some embodiments the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, micro-cystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pectin, andtragacanth. In some embodiments the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the composition is a food product (e.g., a food orbeverage) such as a health food or beverage, a food or beverage forinfants, a food or beverage for pregnant women, athletes, seniorcitizens or other specified group, a functional food, a beverage, a foodor beverage for specified health use, a dietary supplement, a food orbeverage for patients, or an animal feed. Specific examples of the foodsand beverages include various beverages such as juices, refreshingbeverages, tea beverages, drink preparations, jelly beverages, andfunctional beverages; alcoholic beverages such as beers;carbohydrate-containing foods such as rice food products, noodles,breads, and pastas; paste products such as fish hams, sausages, pasteproducts of seafood; retort pouch products such as curries, food dressedwith a thick starchy sauces, and Chinese soups; soups; dairy productssuch as milk, dairy beverages, ice creams, cheeses, and yogurts;fermented products such as fermented soybean pastes, yogurts, fermentedbeverages, and pickles; bean products; various confectionery products,including biscuits, cookies, and the like, candies, chewing gums,gummies, cold desserts including jellies, cream caramels, and frozendesserts; instant foods such as instant soups and instant soy-beansoups; microwavable foods; and the like. Further, the examples alsoinclude health foods and beverages prepared in the forms of powders,granules, tablets, capsules, liquids, pastes, and jellies.

In some embodiments the composition is a food product for animals,including humans. The animals, other than humans, are not particularlylimited, and the composition can be used for various livestock, poultry,pets, experimental animals, and the like. Specific examples of theanimals include pigs, cattle, horses, sheep, goats, chickens, wildducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice,rats, monkeys, and the like, but the animals are not limited thereto.

Therapeutic Agents

In certain aspects, the methods provided herein include theadministration to a subject of a pharmaceutical composition describedherein either alone or in combination with an additional therapeutic. Insome embodiments, the additional therapeutic is an immunosuppressant, asteroid, cancer therapeutic.

In some embodiments the EV is administered to the subject before thetherapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hoursbefore or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 daysbefore). In some embodiments the EV is administered to the subject afterthe therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hoursafter or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after). Insome embodiments, the EV and the therapeutic are administered to thesubject simultaneously or nearly simultaneously (e.g., administrationsoccur within an hour of each other). In some embodiments, the subject isadministered an antibiotic before the EV is administered to the subject(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 days before),In some embodiments, the subjectis administered an antibiotic after the EV is administered to thesubject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 days after). In some embodiments, theEV and the antibiotic are administered to the subject simultaneously ornearly simultaneously (e.g., administrations occur within an hour ofeach other).

In some embodiments, the additional therapeutic is a cancer therapeutic.In some embodiments, the cancer therapeutic is a chemotherapeutic agent.Examples of such chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammall and calicheamicin omegal 1; dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

In some embodiments, the cancer therapeutic is a cancer immunotherapyagent. Immunotherapy refers to a treatment that uses a subject's immunesystem to treat cancer, e.g., checkpoint inhibitors, cancer vaccines,cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.Non-limiting examples of immunotherapies are checkpoint inhibitorsinclude Nivolumab (BMS, anti-PD-1), Pembrolizumab (Merck, anti-PD-1),Ipilimumab (BMS, anti-CTLA-4), MEDI4736 (AstraZeneca, anti-PD-L1), andMPDL3280A (Roche, anti-PD-L1). Other immunotherapies may be tumorvaccines, such as Gardail, Cervarix, BCG, sipulencel-T, Gp100:209-217,AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak,Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901,POL-103A, Belagenpumatucel-L, GSK1572932A, MDX-1279, GV1001, andTecemotide. Immunotherapy may be administered via injection (e.g.,intravenously, intratumorally, subcutaneously, or into lymph nodes), butmay also be administered orally, topically, or via aerosol.Immunotherapies may comprise adjuvants such as cytokines.

In some embodiments, the immunotherapy agent is an immune checkpointinhibitor. Immune checkpoint inhibition broadly refers to inhibiting thecheckpoints that cancer cells can produce to prevent or downregulate animmune response. Examples of immune checkpoint proteins include, but arenot limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA,KIR, LAG3, TIM-3 or VISTA. Immune checkpoint inhibitors can beantibodies or antigen binding fragments thereof that bind to and inhibitan immune checkpoint protein. Examples of immune checkpoint inhibitorsinclude, but are not limited to, nivolumab, pembrolizumab, pidilizumab,AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736,MSB-0020718C, AUR-012 and STI-A1010.

In some embodiments, the methods provided herein include theadministration of a pharmaceutical composition described herein incombination with one or more additional therapeutic. In someembodiments, the methods disclosed herein include the administration oftwo additional immunotherapy agents (e.g., immune checkpoint inhibitor).For example, the methods provided herein include the administration of apharmaceutical composition described herein in combination with a PD-1inhibitor and a CLTA-4 inhibitor or a PD-L1 inhibitor and a CTLA-4inhibitor.

In some embodiments, the immunotherapy agent is an antibody or antigenbinding fragment thereof that, for example, binds to a cancer-associatedantigen. Examples of cancer-associated antigens include, but are notlimited to, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4,alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABLfusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA,carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen(“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1,G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV,gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11,HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein,Lengsin, M-CSF, MAGE-A1 MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2,MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2,MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9,P polypeptide, p53, PAP, PAXS, PBF, pml-RARalpha fusion protein,polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDXS, PSA, PSMA,PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGSS, RhoC, RNF43, RU2AS, SAGE,secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1,survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase,TGF-betaRll, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2,TRP2-INT2, tyrosinase, tyrosinase (“TYR”), dVEGF, WT1, XAGE-1b/GAGED2a.In some embodiments, the antigen is a neo-antigen.

In some embodiments, the immunotherapy agent is a cancer vaccine and/ora component of a cancer vaccine (e.g., an antigenic peptide and/orprotein). The cancer vaccine can be a protein vaccine, a nucleic acidvaccine or a combination thereof. For example, in some embodiments, thecancer vaccine comprises a polypeptide comprising an epitope of acancer-associated antigen. In some embodiments, the cancer vaccinecomprises a nucleic acid (e.g., DNA or RNA, such as mRNA) that encodesan epitope of a cancer-associated antigen. Examples of cancer-associatedantigens include, but are not limited to, adipophilin, AIM-2, ALDH1A1,alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA,carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,cyclin D1, Cyclin-A1,dek-can fusion protein, DKK1, EFTUD2, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen(“ETA”), ETV6-AML1 fusion protein, EZH2, FGFS, FLT3-ITD, FN1,G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV,gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11,HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1also known as CCDC110, LAGE-1, LDLR-fucosyltranferaseAS fusion protein,Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2,MATN, MC1R, MCSP, mdm-2, MEL Melan-A/MART-1, Meloe, Midkine, MMP-2,MMP-7, MUC1, MUCSAC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9,P polypeptide, p53, PAP, PAXS, PBF, pm1-RARalpha fusion protein,polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDXS, PSA, PSMA,PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGSS, RhoC, RNF43, RU2AS, SAGE,secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1,survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase,TGF-betaRll, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2,TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a.In some embodiments, the antigen is a neo-antigen. In some embodiments,the cancer vaccine is administered with an adjuvant. Examples ofadjuvants include, but are not limited to, an immune modulatory protein,Adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calciumphosphate, β-Glucan Peptide, CpG ODN DNA, GPI-0100, lipid A,lipopolysaccharide, Lipovant, Montanide,N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A , choleratoxin (CT) and heat-labile toxin from enterotoxigenic Escherichia coli(LT) including derivatives of these (CTB, mmCT, CTA1 -DD, LTB, LTK63,LTR72, dmLT) and trehalose dimycolate.

In some embodiments, the immunotherapy agent is an immune modulatingprotein to the subject. In some embodiments, the immune modulatoryprotein is a cytokine or chemokine. Examples of immune modulatingproteins include, but are not limited to, B lymphocyte chemoattractant(“BLC”), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotacticprotein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor(“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”),1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon alpha(“IFN-alpha”), Interferon beta (“IFN-beta”) Interferon gamma(“IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta(“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1 ra”),Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”),Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Subunit beta of Interleukin-12(“IL-12 p40” or “IL-12 p70”), Interleukin-13 (“IL-13”), Interleukin-15(“IL-15”), Interleukin-16 (“IL-16”), Interleukin-17A-F (“IL-17A-F”),Interleukin-18 (“IL-18”), Interleukin-21 (“IL-21”), Interleukin-22(“IL-22”), Interleukin-23 (“IL-23”), Interleukin-33 (“IL-33”), Chemokine(C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor(“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-Cmotif) ligand 2 (“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 (“MIP-1beta”), Macrophage inflammatory protein-1 -delta (“MIP-1 delta”),Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-Cmotif) ligand 5, Regulated on Activation, Normal T cell Expressed andSecreted (“RANTES”), TIMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMPmetallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor,lymphotoxin-alpha (“TNF alpha”), Tumor necrosis factor, lymphotoxin-beta(“TNF beta”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR,Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growthfactor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bonemorphogenetic protein 5 ('BMP-5″), Bone morphogenetic protein 7('BMP-7″), Nerve growth factor (“b-NGF”), Epidermal growth factor(“EGF”), Epidermal growth factor receptor (“EGFR”),Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”),Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor(“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glialcell-derived neurotrophic factor (“GDNF”), Growth Hormone,Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growthfactor (“HGF”), Insulin-like growth factor binding protein 1(“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”),Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-likegrowth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factorbinding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”),Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growthfactor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4(“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”),Platelet-derived growth factor receptors (“PDGF-AA”),Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-boxcontaining comples (“SCF”), Stem cell factor receptor (“SCF R”),Transforming growth factor alpha (“TGFalpha”), Transforming growthfactor beta-1 (“TGF beta 1”), Transforming growth factor beta-3 (“TGFbeta 3”), Vascular endothelial growth factor (“VEGF”), Vascularendothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelialgrowth factor receptor 3 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-proteinkinase receptor UFO (“Axl”) , Betacellulin (“BTC”), Mucosae-associatedepithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27(“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motifchemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”),Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif)ligand 14 (“HCC-1”), Chemokine (C-C motif) ligand 16 (“HCC-4”),Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”), Interleukin-18-binding protein (“IL-18 BPa”), Interleukin-28 A (“IL-28A”),Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C-X-C motifchemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”), Leukemiainhibitory factor (“LIF”), Light, Chemokine (C motif) ligand(“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocytechemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4(“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migrationinhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3alpha”), C-C motif chemokine 19 (“MIP-3 beta”), Chemokine (C-C motif)ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain(“MSPalpha”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secretedphosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulatedcytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derivedfactor- 1 alpha (“SDF-1 alpha”), Chemokine (C-C motif) ligand 17(“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromallymphopoietin (“TSLP 4- IBB”), CD 166 antigen (“ALCAM”), Cluster ofDifferentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamilymember 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster ofDifferentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1(biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”),Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein(“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”),Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosisantigen 1(“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosisfactor receptor superfamily member 1 (“GITR”), Tumor necrosis factorreceptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rbeta, IL-17R, IL-2Rgamma, IL-21R,Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associatedlipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium(“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHCclass I polypeptide-related sequence B (“MICB”), NRG1-betal, Beta-typeplatelet-derived growth factor receptor (“PDGF Rbeta”), Plateletendothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A viruscellular receptor 1 (“TIM-1”), Tumor necrosis factor receptorsuperfamily member IOC (“TRAIL R3”), Trappin protein transglutaminasebinding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular celladhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein(“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin,Catheprin S, CD40, Cryptic family protein IB (“Cripto-1”), DAN,Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial celladhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C,FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”),IL-13 R1, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal celladhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1(“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”),Resistin, stromal cell-derived factor 1 (“SDF-1 beta”), sgp130, Secretedfrizzled-related protein 2 (“ShhN”), Sialic acid-bindingimmunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growthfactor-beta 2 (“TGF beta 2”), Tie-2, Thrombopoietin (“TPO”), Tumornecrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggeringreceptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelialgrowth factor C (“VEGF-C”), VEGFR1Adiponectin, Adipsin (“AND”),Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”),Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”),Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”),Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), HumanEpidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin,Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1(“GRO alpha”), human chorionic gonadotropin (“beta HCG”), Insulin-likegrowth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21,Leptin, Matrix metalloproteinase-1 (“MMP-1”), Matrix metalloproteinase-2(“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrixmetalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”),Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13(“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin(“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”),Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialicacid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase(“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”),TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9(“ADAM-9”), Angiopoietin 2, Tumor necrosis factor ligand superfamilymember 13/Acidic leucine-rich nuclear phosphoprotein 32 family member B(“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogeneticprotein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L,CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblastactivation protein, alpha (“FAP”), Fibroblast growth factor 19(“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”),IFN-gammalpha/beta R2, Insulin-like growth factor 2 (“IGF-2”),Insulin-like growth factor 2 receptor (“IGF-2 R”), Interleukin-1receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”), Interleukin 33(“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”),Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-bindinglectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1,translocation-associated (Drosophila) (“Notch-1”), Nephroblastomaoverexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1(“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4,Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Tolllikereceptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member10A (“TRAIL R1”), Transferrin (“TRF”), WIF-1ACE-2, Albumin, AMICA,Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen19-9 (“CA19-9”), CD 163 , Clusterin, CRT AM, Chemokine (C-X-C motif)ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-relatedprotein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A,Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha(“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”),GPCR-associated sorting protein 2 (“GASP-2”), Granulocytecolony-stimulating factor receptor (“GCSF R”), Serine protease hepsin(“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27(“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V(“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST,Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factorreceptor superfamily member 13B (“TACI”), Tissue factor pathwayinhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily,member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase PlasminogenActivator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascularendothelial) also known as CD144 (“VE-Cadherin”),WNT1-inducible-signaling pathway protein 1 (“WISP-1”), and ReceptorActivator of Nuclear Factor κ B (“RANK”).

In some embodiments, the cancer therapeutic agent is an anti-cancercompound. Exemplary anti-cancer compounds include, but are not limitedto, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole(Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib(Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®),Cabozantinib (Cometriq™), Carfilzomib (Kyprolis™), Cetuximab (Erbitux®),Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox(Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afinitor®),Exemestane (Aromasin®), Fulvestrant (Faslodex®), Gefitinib (Iressa®),Ibritumomab tiuxetan (Zevalin®), Imatinib mesylate (Gleevec®),Ipilimumab (Yervoy™), Lapatinib ditosylate (Tykerb®), Letrozole(Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab(Vectibix®), Pazopanib hydrochloride (Votrient®), Pertuzumab (Perjeta™),Pralatrexate (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®),Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate(Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®),Tositumomab and 131I-tositumomab (Bexxar®), Trastuzumab (Herceptin®),Tretinoin (Vesanoid®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®),Vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®).

Exemplary anti-cancer compounds that modify the function of proteinsthat regulate gene expression and other cellular functions (e.g., MACinhibitors, retinoid receptor ligants) are Vorinostat (Zolinza®),Bexarotene (Targretin®) and Romidepsin (Istodax®), Alitretinoin(Panretin®), and Tretinoin (Vesanoid®).

Exemplary anti-cancer compounds that induce apoptosis (e.g., proteasomeinhibitors, antifolates) are Bortezomib (Velcade®), Carfilzomib(Kyprolis™), and Pralatrexate (Folotyn®).

Exemplary anti-cancer compounds that increase anti-tumor immune response(e.g., anti CD20, anti CD52; anti-cytotoxic T-lymphocyte-associatedantigen-4) are Rituximab (Rituxan®), Alemtuzumab (Campath®), Ofatumumab(Arzerra®), and Ipilimumab (Yervoy™)

Exemplary anti-cancer compounds that deliver toxic agents to cancercells (e.g., anti-CD20-radionuclide fusions; IL-2-diphtheria toxinfusions; anti-CD30- monomethylauristatin E (MMAE)-fusions) areTositumomab and 1311-tositumomab (Bexxar®)and Ibritumomab tiuxetan(Zevalin®), Denileukin diftitox (Ontak®), and Brentuximab vedotin(Adcetris®).

Other exemplary anti-cancer compounds are small molecule inhibitors andconjugates thereof of, e.g., Janus kinase, ALK, Bcl-2, PARP, PI3K, VEGFreceptor, Braf, MEK, CDK, and HSP90.

Exemplary platinum-based anti-cancer compounds include, for example,cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,Nedaplatin, Triplatin, and Lipoplatin. Other metal-based drugs suitablefor treatment include, but are not limited to ruthenium-based compounds,ferrocene derivatives, titanium-based compounds, and gallium-basedcompounds.

In some embodiments, the cancer therapeutic is a radioactive moiety thatcomprises a radionuclide. Exemplary radionuclides include, but are notlimited to Cr-51, Cs-131, Ce-134, Se-75, Ru-97, 1-125, Eu-149, Os-189m,Sb-119, 1-123, Ho-161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, T1-201,Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P-33, Er-169, Ru-103, Yb-169,Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu-177, Rh-105,Sn-117m, Cu-67, Sc-47, Pt-195m, Ce-141, 1-131, Tb-161, As-77, Pt-197,Sm-153, Gd-159, Tm-173, Pr-143, Au-198, Tm-170, Re-186, Ag-111, Pd-109,Ga-73, Dy-165, Pm-149, Sn-123, Sr-89, Ho-166, P-32, Re-188, Pr-142,Ir-194, In-114m/In-114, and Y-90.

In some embodiments, the cancer therapeutic is an antibiotic. Forexample, if the presence of a cancer-associated bacteria and/or acancer-associated microbiome profile is detected according to themethods provided herein, antibiotics can be administered to eliminatethe cancer-associated bacteria from the subject. “Antibiotics” broadlyrefers to compounds capable of inhibiting or preventing a bacterialinfection. Antibiotics can be classified in a number of ways, includingtheir use for specific infections, their mechanism of action, theirbioavailability, or their spectrum of target microbe (e.g.,Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobicbacteria, etc.) and these may be used to kill specific bacteria inspecific areas of the host (“niches”) (Leekha, et al 2011. GeneralPrinciples of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167). Incertain embodiments, antibiotics can be used to selectively targetbacteria of a specific niche. In some embodiments, antibiotics known totreat a particular infection that includes a cancer niche may be used totarget cancer-associated microbes, including cancer-associated bacteriain that niche. In other embodiments, antibiotics are administered afterthe bacterial treatment. In some embodiments, antibiotics areadministered after the bacterial treatment to remove the engraftment.

In some aspects, antibiotics can be selected based on their bactericidalor bacteriostatic properties. Bactericidal antibiotics includemechanisms of action that disrupt the cell wall (e.g., β-lactams), thecell membrane (e.g., daptomycin), or bacterial DNA (e.g.,fluoroquinolones). Bacteriostatic agents inhibit bacterial replicationand include sulfonamides, tetracyclines, and macrolides, and act byinhibiting protein synthesis. Furthermore, while some drugs can bebactericidal in certain organisms and bacteriostatic in others, knowingthe target organism allows one skilled in the art to select anantibiotic with the appropriate properties. In certain treatmentconditions, bacteriostatic antibiotics inhibit the activity ofbactericidal antibiotics. Thus, in certain embodiments, bactericidal andbacteriostatic antibiotics are not combined.

Antibiotics include, but are not limited to aminoglycosides, ansamycins,carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides,lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones,penicillins, polypeptide antibiotics, quinolones, fluoroquinolone,sulfonamides, tetracyclines, and anti-mycobacterial compounds, andcombinations thereof.

Aminoglycosides include, but are not limited to Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, andSpectinomycin. Aminoglycosides are effective, e.g., againstGram-negative bacteria, such as Escherichia coli, Klebsiella,Pseudomonas aeruginosa, and Francisella tularensis, and against certainaerobic bacteria but less effective against obligate/facultativeanaerobes. Aminoglycosides are believed to bind to the bacterial 30S or50S ribosomal subunit thereby inhibiting bacterial protein synthesis.

Ansamycins include, but are not limited to, Geldanamycin, Herbimycin,Rifamycin, and Streptovaricin. Geldanamycin and Herbimycin are believedto inhibit or alter the function of Heat Shock Protein 90.

Carbacephems include, but are not limited to, Loracarbef. Carbacephemsare believed to inhibit bacterial cell wall synthesis.

Carbapenems include, but are not limited to, Ertapenem, Doripenem,Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal forboth Gram-positive and Gram-negative bacteria as broad-spectrumantibiotics. Carbapenems are believed to inhibit bacterial cell wallsynthesis.

Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin,Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin,Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftriaxone, Cefepime, Ceftaroline fosamil,and Ceftobiprole. SelectedCephalosporins are effective, e.g., against Gram-negative bacteria andagainst Gram-positive bacteria, including Pseudomonas, certainCephalosporins are effective against methicillin-resistantStaphylococcus aureus (MRSA). Cephalosporins are believed to inhibitbacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin,and Telavancin. Glycopeptides are effective, e.g., against aerobic andanaerobic Gram-positive bacteria including MRSA and Clostridiumdifficile. Glycopeptides are believed to inhibit bacterial cell wallsynthesis by disrupting synthesis of the peptidoglycan layer ofbacterial cell walls.

Lincosamides include, but are not limited to, Clindamycin andLincomycin. Lincosamides are effective, e.g., against anaerobicbacteria, as well as Staphylococcus, and Streptococcus. Lincosamides arebelieved to bind to the bacterial 50S ribosomal subunit therebyinhibiting bacterial protein synthesis.

Lipopeptides include, but are not limited to, Daptomycin. Lipopeptidesare effective, e.g., against Gram-positive bacteria. Lipopeptides arebelieved to bind to the bacterial membrane and cause rapiddepolarization.

Macrolides include, but are not limited to, Azithromycin,Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective,e.g., against Streptococcus and Mycoplasma. Macrolides are believed tobind to the bacterial or 50S ribosomal subunit, thereby inhibitingbacterial protein synthesis.

Monobactams include, but are not limited to, Aztreonam. Monobactams areeffective, e.g., against Gram-negative bacteria. Monobactams arebelieved to inhibit bacterial cell wall synthesis by disruptingsynthesis of the peptidoglycan layer of bacterial cell walls.

Nitrofurans include, but are not limited to, Furazolidone andNitrofurantoin.

Oxazolidonones include, but are not limited to, Linezolid, Posizolid,Radezolid, and Torezolid. Oxazolidonones are believed to be proteinsynthesis inhibitors.

Penicillins include, but are not limited to, Amoxicillin, Ampicillin,Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin,Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, PenicillinV, Piperacillin, Temocillin and Ticarcillin. Penicillins are effective,e.g., against Gram-positive bacteria, facultative anaerobes, e.g.,Streptococcus, Borrelia, and Treponema. Penicillins are believed toinhibit bacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Penicillin combinations include, but are not limited to,Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,and Ticarcillin/clavulanate.

Polypeptide antibiotics include, but are not limited to, Bacitracin,Colistin, and Polymyxin B and E. Polypeptide Antibiotics are effective,e.g., against Gram-negative bacteria. Certain polypeptide antibioticsare believed to inhibit isoprenyl pyrophosphate involved in synthesis ofthe peptidoglycan layer of bacterial cell walls, while othersdestabilize the bacterial outer membrane by displacing bacterialcounter-ions.

Quinolones and Fluoroquinolone include, but are not limited to,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.Quinolones/Fluoroquinolone are effective, e.g., against Streptococcusand Neisseria. Quinolones/Fluoroquinolone are believed to inhibit thebacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNAreplication and transcription.

Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide,Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole,Sulfamethoxazole,

Sulfanilimide, Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole (Co-trimoxazole), andSulfonamidochrysoidine. Sulfonamides are believed to inhibit folatesynthesis by competitive inhibition of dihydropteroate synthetase,thereby inhibiting nucleic acid synthesis.

Tetracyclines include, but are not limited to, Demeclocycline,Doxycycline, Minocycline, Oxytetracycline, and Tetracycline.Tetracyclines are effective, e.g., against Gram-negative bacteria.Tetracyclines are believed to bind to the bacterial 30S ribosomalsubunit thereby inhibiting bacterial protein synthesis.

Anti-mycobacterial compounds include, but are not limited to,Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide,Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, andStreptomycin.

Suitable antibiotics also include arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprimamoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin,azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl,clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate,gramicidin, imipenem, indolicidin, josamycin, magainan II,metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacinB-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleandomycin,ostreogrycin, piperacillin/tazobactam, pristinamycin, ramoplanin,ranalexin, reuterin, rifaximin, rosamicin, rosaramicin, spectinomycin,spiramycin, staphylomycin, streptogramin, streptogramin A, synergistin,taurolidine, teicoplanin, telithromycin, ticarcillin/clavulanic acid,triacetyloleandomycin, tylosin, tyrocidin, tyrothricin, vancomycin,vemamycin, and virginiamycin.

In some embodiments, the additional therapeutic is an immunosuppressiveagent, a DMARD, a pain-control drug, a steroid, a non-steroidalantiinflammatory drug (NSAID), or a cytokine antagonist, andcombinations thereof. Representative agents include, but are not limitedto, cyclosporin, retinoids, corticosteroids, propionic acid derivative,acetic acid derivative, enolic acid derivatives, fenamic acidderivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesiumsalicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen,flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac,nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib,acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamicacid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac,indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX),antimalarial drugs (e.g., hydroxychloroquine and chloroquine),sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts,minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin,tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists (e.g., TNFalpha antagonists or TNF alpha receptor antagonists), e.g., ADALIMUMAB(Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650),CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148),ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT(Orencia®), TOCILIZUMAB (RoActemra /Actemra®), integrin antagonists(TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Ilaris)), Anakinra(Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6antagonists, BLyS antagonists (e.g., Atacicept, Benlysta®/ LymphoStat-B®(belimumab)), p38 Inhibitors, CD20 antagonists (Ocrelizumab, Ofatumumab(Arzerra®)), interferon gamma antagonists (Fontolizumab), prednisolone,Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone,methylprednisolone, betamethasone, triamcinolone, beclometasome,fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline,vancomycin, pioglitazone, SBI-087, SC10-469, Cura-100, Oncoxin+Viusid,TwHF, Methoxsalen, Vitamin D-ergocalciferol, Milnacipran, Paclitaxel,rosig tazone, Tacrolimus (Prograf®), RADOO1, rapamune, rapamycin,fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium,rosightazone,Curcumin (Longvida™) Rosuvastatin, Maraviroc, ramipnl, Milnacipran,Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553,esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAKinhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980,denosumab, IL-6 antagonists, CD20 antagonistis, CTLA4 antagonists, IL-8antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists(Tysarbri® (natalizumab)), VGEF antagnosits, CXCL antagonists, MMPantagonists, defensin antagonists, IL-1 antagonists (including IL-1 betaantagonsits), and IL-23 antagonists (e.g., receptor decoys, antagonisticantibodies, etc.).

In some embodiments, the agent is an immunosuppressive agent. Examplesof immunosuppressive agents include, but are not limited to,corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazinederivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine,azathiopurine, prednisone, methotrexate, antihistamines,glucocorticoids, epinephrine, theophylline, cromolyn sodium,anti-leukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists,inflammasome inhibitors, anti-cholinergic decongestants, mast-cellstabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccinesused for vaccination where the amount of an allergen is graduallyincreased), cytokine inhibitors, such as anti-IL-6 antibodies, TNFinhibitors such as infliximab, adalimumab, certolizumab pegol,golimumab, or etanercept, iand combinations thereof.

Administration

In certain aspects, provided herein is a method of delivering apharmaceutical composition described herein to a subject. In someembodiments of the methods provided herein, the pharmaceuticalcomposition is administered in conjunction with the administration of anadditional therapeutic. In some embodiments, the pharmaceuticalcomposition comprises EVs and/or bacteria co-formulated with theadditional therapeutic. In some embodiments, the pharmaceuticalcomposition is co-administered with the additional therapeutic. In someembodiments, the additional therapeutic is administered to the subjectbefore administration of the pharmaceutical composition (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutesbefore, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 days before). In some embodiments, theadditional therapeutic is administered to the subject afteradministration of the pharmaceutical composition (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutesafter, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 days after). In some embodiments the same modeof delivery are used to deliver both the pharmaceutical composition andthe additional therapeutic. In some embodiments different modes ofdelivery are used to administer the pharmaceutical composition and theadditional therapeutic. For example, in some embodiments thepharmaceutical composition is administered orally while the additionaltherapeutic is administered via injection (e.g., an intravenous,intramuscular and/or intratumoral injection).

In certain embodiments, the pharmaceutical compositions, dosage forms,and kits described herein can be administered in conjunction with anyother conventional anti-cancer treatment, such as, for example,radiation therapy and surgical resection of the tumor. These treatmentsmay be applied as necessary and/or as indicated and may occur before,concurrent with or after administration of the pharmaceuticalcompositions, dosage forms, and kits described herein.

The dosage regimen can be any of a variety of methods and amounts, andcan be determined by one skilled in the art according to known clinicalfactors. As is known in the medical arts, dosages for any one patientcan depend on many factors, including the subject's species, size, bodysurface area, age, sex, immunocompetence, and general health, theparticular microorganism to be administered, duration and route ofadministration, the kind and stage of the disease, for example, tumorsize, and other compounds such as drugs being administered concurrently.In addition to the above factors, such levels can be affected by theinfectivity of the microorganism, and the nature of the microorganism,as can be determined by one skilled in the art. In the present methods,appropriate minimum dosage levels of microorganisms can be levelssufficient for the microorganism to survive, grow and replicate. Thedose of the pharmaceutical compositions described herein may beappropriately set or adjusted in accordance with the dosage form, theroute of administration, the degree or stage of a target disease, andthe like. For example, the general effective dose of the agents mayrange between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day,between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg bodyweight/day and 100 mg/kg body weight/day, or between 5 mg/kg bodyweight/day and 50 mg/kg body weight/day. The effective dose may be 0.01,0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 500, or 1000 mg/kg body weight/day or more, but the dose is notlimited thereto.

In some embodiments, the dose administered to a subject is sufficient toprevent disease (e.g., autoimmune disease, inflammatory disease,metabolic disease, cancer), delay its onset, or slow or stop itsprogression. One skilled in the art will recognize that dosage willdepend upon a variety of factors including the strength of theparticular compound employed, as well as the age, species, condition,and body weight of the subject. The size of the dose will also bedetermined by the route, timing, and frequency of administration as wellas the existence, nature, and extent of any adverse side-effects thatmight accompany the administration of a particular compound and thedesired physiological effect.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. An effective dosage and treatment protocol canbe determined by routine and conventional means, starting e.g., with alow dose in laboratory animals and then increasing the dosage whilemonitoring the effects, and systematically varying the dosage regimen aswell. Animal studies are commonly used to determine the maximaltolerable dose (“MTD”) of bioactive agent per kilogram weight. Thoseskilled in the art regularly extrapolate doses for efficacy, whileavoiding toxicity, in other species, including humans.

In accordance with the above, in therapeutic applications, the dosagesof the active agents used in accordance with the invention varydepending on the active agent, the age, weight, and clinical conditionof the recipient patient, and the experience and judgment of theclinician or practitioner administering the therapy, among other factorsaffecting the selected dosage. Generally, the dose should be sufficientto result in slowing, and preferably regressing, the growth of thetumors and most preferably causing complete regression of the cancer.

Separate administrations can include any number of two or moreadministrations, including two, three, four, five or sixadministrations. One skilled in the art can readily determine the numberof administrations to perform or the desirability of performing one ormore additional administrations according to methods known in the artfor monitoring therapeutic methods and other monitoring methods providedherein. Accordingly, the methods provided herein include methods ofproviding to the subject one or more administrations of anpharmaceutical composition, where the number of administrations can bedetermined by monitoring the subject, and, based on the results of themonitoring, determining whether or not to provide one or more additionaladministrations. Deciding on whether or not to provide one or moreadditional administrations can be based on a variety of monitoringresults.

The time period between administrations can be any of a variety of timeperiods. The time period between administrations can be a function ofany of a variety of factors, including monitoring steps, as described inrelation to the number of administrations, the time period for a subjectto mount an immune response and/or the time period for a subject toclear the EV from normal tissue. In one example, the time period can bea function of the time period for a subject to mount an immune response;for example, the time period can be more than the time period for asubject to mount an immune response, such as more than about one week,more than about ten days, more than about two weeks, or more than abouta month; in another example, the time period can be less than the timeperiod for a subject to mount an immune response, such as less thanabout one week, less than about ten days, less than about two weeks, orless than about a month. In another example, the time period can be afunction of the time period for a subject to clear the EV from normaltissue; for example, the time period can be more than the time periodfor a subject to clear the EV from normal tissue, such as more thanabout a day, more than about two days, more than about three days, morethan about five days, or more than about a week.

In some embodiments, the delivery of an additional therapeutic incombination with the pharmaceutical composition described herein reducesthe adverse effects and/or improves the efficacy of the additionaltherapeutic.

The effective dose of an additional therapeutic described herein is theamount of the therapeutic agent that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, with the least toxicity to the patient. The effectivedosage level can be identified using the methods described herein andwill depend upon a variety of pharmacokinetic factors including theactivity of the particular compositions administered, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. In general, aneffective dose of an additional therapy will be the amount of thetherapeutic agent which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above.

The toxicity of an additional therapy is the level of adverse effectsexperienced by the subject during and following treatment. Adverseevents associated with additional therapy toxicity include, but are notlimited to, abdominal pain, acid indigestion, acid reflux, allergicreactions, alopecia, anaphylasix, anemia, anxiety, lack of appetite,arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain,bleeding, blood clots, low blood pressure, elevated blood pressure,difficulty breathing, bronchitis, bruising, low white blood cell count,low red blood cell count, low platelet count, cardiotoxicity, cystitis,hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy,coronary artery disease, cataracts, central neurotoxicity, cognitiveimpairment, confusion, conjunctivitis, constipation, coughing, cramping,cystitis, deep vein thrombosis, dehydration, depression, diarrhea,dizziness, dry mouth, dry skin, dyspepsia, dyspnea, edema, electrolyteimbalance, esophagitis, fatigue, loss of fertility, fever, flatulence,flushing, gastric reflux, gastroesophageal reflux disease, genital pain,granulocytopenia, gynecomastia, glaucoma, hair loss, hand-foot syndrome,headache, hearing loss, heart failure, heart palpitations, heartburn,hematoma, hemorrhagic cystitis, hepatotoxicity, hyperamylasemia,hypercalcemia, hyperchloremia, hyperglycemia, hyperkalemia,hyperlipasemia, hypermagnesemia, hypernatremia, hyperphosphatemia,hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia,hypocalcemia, hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia,hyponatremia, hypophosphatemia, impotence, infection, injection sitereactions, insomnia, iron deficiency, itching, joint pain, kidneyfailure, leukopenia, liver dysfunction, memory loss, menopause, mouthsores, mucositis, muscle pain, myalgias, myelosuppression, myocarditis,neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds,numbness, ototoxicity, pain, palmar-plantar erythrodysesthesia,pancytopenia, pericarditis, peripheral neuropathy, pharyngitis,photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria,pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapidheart beat, rectal bleeding, restlessness, rhinitis, seizures, shortnessof breath, sinusitis, thrombocytopenia, tinnitus, urinary tractinfection, vaginal bleeding, vaginal dryness, vertigo, water retention,weakness, weight loss, weight gain, and xerostomia. In general, toxicityis acceptable if the benefits to the subject achieved through thetherapy outweigh the adverse events experienced by the subject due tothe therapy.

Immune Disorders

In some embodiments, the methods and compositions described hereinrelate to the treatment or prevention a disease or disorder associated apathological immune response, such as an autoimmune disease, an allergicreaction and/or an inflammatory disease. In some embodiments, thedisease or disorder is an inflammatory bowel disease (e.g., Crohn'sdisease or ulcerative colitis).

The methods described herein can be used to treat any subject in needthereof. As used herein, a “subject in need thereof” includes anysubject that has a disease or disorder associated with a pathologicalimmune response (e.g., an inflammatory bowel disease), as well as anysubject with an increased likelihood of acquiring a such a disease ordisorder.

The compositions described herein can be used, for example, as apharmaceutical composition for preventing or treating (reducing,partially or completely, the adverse effects of) an autoimmune disease,such as chronic inflammatory bowel disease, systemic lupuserythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis,multiple sclerosis, or Hashimoto's disease; an allergic disease, such asa food allergy, pollenosis, or asthma; an infectious disease, such as aninfection with Clostridium difficile; an inflammatory disease such as aTNF-mediated inflammatory disease (e.g., an inflammatory disease of thegastrointestinal tract, such as pouchitis, a cardiovascular inflammatorycondition, such as atherosclerosis, or an inflammatory lung disease,such as chronic obstructive pulmonary disease); a pharmaceuticalcomposition for suppressing rejection in organ transplantation or othersituations in which tissue rejection might occur; a supplement, food, orbeverage for improving immune functions; or a reagent for suppressingthe proliferation or function of immune cells.

In some embodiments, the methods provided herein are useful for thetreatment of inflammation. In certain embodiments, the inflammation ofany tissue and organs of the body, including musculoskeletalinflammation, vascular inflammation, neural inflammation, digestivesystem inflammation, ocular inflammation, inflammation of thereproductive system, and other inflammation, as discussed below.

Immune disorders of the musculoskeletal system include, but are notlimited, to those conditions affecting skeletal joints, including jointsof the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle,and foot, and conditions affecting tissues connecting muscles to bonessuch as tendons. Examples of such immune disorders, which may be treatedwith the methods and compositions described herein include, but are notlimited to, arthritis (including, for example, osteoarthritis,rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acuteand chronic infectious arthritis, arthritis associated with gout andpseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis,tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis,myositis, and osteitis (including, for example, Paget's disease,osteitis pubis, and osteitis fibrosa cystic).

Ocular immune disorders refers to a immune disorder that affects anystructure of the eye, including the eye lids. Examples of ocular immunedisorders which may be treated with the methods and compositionsdescribed herein include, but are not limited to, blepharitis,blepharochalasis, conjunctivitis, dacryoadenitis, keratitis,keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis

Examples of nervous system immune disorders which may be treated withthe methods and compositions described herein include, but are notlimited to, encephalitis, Guillain-Barre syndrome, meningitis,neuromyotonia, narcolepsy, multiple sclerosis, myelitis andschizophrenia. Examples of inflammation of the vasculature or lymphaticsystem which may be treated with the methods and compositions describedherein include, but are not limited to, arthrosclerosis, arthritis,phlebitis, vasculitis, and lymphangitis.

Examples of digestive system immune disorders which may be treated withthe methods and compositions described herein include, but are notlimited to, cholangitis, cholecystitis, enteritis, enterocolitis,gastritis, gastroenteritis, inflammatory bowel disease, ileitis, andproctitis. Inflammatory bowel diseases include, for example, certainart-recognized forms of a group of related conditions. Several majorforms of inflammatory bowel diseases are known, with Crohn's disease(regional bowel disease, e.g., inactive and active forms) and ulcerativecolitis (e.g., inactive and active forms) the most common of thesedisorders. In addition, the inflammatory bowel disease encompassesirritable bowel syndrome, microscopic colitis, lymphocytic-plasmocyticenteritis, coeliac disease, collagenous colitis, lymphocytic colitis andeosinophilic enterocolitis. Other less common forms of IBD includeindeterminate colitis, pseudomembranous colitis (necrotizing colitis),ischemic inflammatory bowel disease, Behcet's disease, sarcoidosis,scleroderma, IBD-associated dysplasia, dysplasia associated masses orlesions, and primary sclerosing cholangitis.

Examples of reproductive system immune disorders which may be treatedwith the methods and compositions described herein include, but are notlimited to, cervicitis, chorioamnionitis, endometritis, epididymitis,omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess,urethritis, vaginitis, vulvitis, and vulvodynia.

The methods and compositions described herein may be used to treatautoimmune conditions having an inflammatory component. Such conditionsinclude, but are not limited to, acute disseminated alopeciauniversalise, Behcet's disease, Chagas' disease, chronic fatiguesyndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis,aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis,autoimmune oophoritis, celiac disease, Crohn's disease, diabetesmellitus type 1, giant cell arteritis, goodpasture's syndrome, Grave'sdisease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonleinpurpura, Kawasaki's disease, lupus erythematosus, microscopic colitis,microscopic polyarteritis, mixed connective tissue disease, Muckle-Wellssyndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonussyndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritisnodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren'ssyndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmunehaemolytic anemia, interstitial cystitis, Lyme disease, morphea,psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

The methods and compositions described herein may be used to treatT-cell mediated hypersensitivity diseases having an inflammatorycomponent. Such conditions include, but are not limited to, contacthypersensitivity, contact dermatitis (including that due to poison ivy),uticaria, skin allergies, respiratory allergies (hay fever, allergicrhinitis, house dustmite allergy) and gluten-sensitive enteropathy(Celiac disease).

Other immune disorders which may be treated with the methods andcompositions include, for example, appendicitis, dermatitis,dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis,hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis,myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis,peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis,pyelonephritis, and stomatisi, transplant rejection (involving organssuch as kidney, liver, heart, lung, pancreas (e.g., islet cells), bonemarrow, cornea, small bowel, skin allografts, skin homografts, and heartvalve xengrafts, sewrum sickness, and graft vs host disease), acutepancreatitis, chronic pancreatitis, acute respiratory distress syndrome,Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurativethyroiditis, hypercalcemia associated with cancer, pemphigus, bullousdermatitis herpetiformis, severe erythema multiforme, exfoliativedermatitis, seborrheic dermatitis, seasonal or perennial allergicrhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drughypersensistivity reactions, allergic conjunctivitis, keratitis, herpeszoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, opticneuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonarytuberculosis chemotherapy, idiopathic thrombocytopenic purpura inadults, secondary thrombocytopenia in adults, acquired (autoimmune)haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia ofchildhood, regional enteritis, autoimmune vasculitis, multiplesclerosis, chronic obstructive pulmonary disease, solid organ transplantrejection, sepsis. Preferred treatments include treatment of transplantrejection, rheumatoid arthritis, psoriatic arthritis, multiplesclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemiclupus erythematosus, psoriasis, chronic obstructive pulmonary disease,and inflammation accompanying infectious conditions (e.g., sepsis).

Metabolic Disorders

The methods and compositions described herein may be used to treatmetabolic disorders and metabolic syndromes. Such conditions include,but are not limited to, Type II Diabetes, Encephalopathy, Tay-Sachsdisease, Krabbe disease, Galactosemia, Phenylketonuria (PKU), and Maplesyrup urine disease. Accordingly, in certain embodiments provided hereinare methods of treating metabolic diseases comprising administering to asubject a composition provided herein. In certain embodiments themetabolic disease is Type II Diabetes, Encephalopathy, Tay-Sachsdisease, Krabbe disease, Galactosemia, Phenylketonuria (PKU), or Maplesyrup urine disease.

Cancer

In some embodiments, the methods and compositions described hereinrelate to the treatment of cancer. In some embodiments, any cancer canbe treated using the methods described herein. Examples of cancers thatmay treated by methods and compositions described herein include, butare not limited to, cancer cells from the bladder, blood, bone, bonemarrow, brain, breast, colon, esophagus, gastrointestine, gum, head,kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach,testis, tongue, or uterus. In addition, the cancer may specifically beof the following histological type, though it is not limited to these:neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant andspindle cell carcinoma; small cell carcinoma; papillary carcinoma;squamous cell carcinoma; lymphoepithelial carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellularcarcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoidcystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;follicular adenocarcinoma; papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma;adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma,malignant; ovarian stromal tumor, malignant; thecoma, malignant;granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cellcarcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;paraganglioma, malignant; extra-mammary paraganglioma, malignant;pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanoticmelanoma; superficial spreading melanoma; malig melanoma in giantpigmented nevus; epithelioid cell melanoma; blue nevus, malignant;sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonalrhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixedtumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.

In some embodiments, the methods and compositions provided herein relateto the treatment of a leukemia. The term “leukemia” is meant broadlyprogressive, malignant diseases of the hematopoietic organs/systems andis generally characterized by a distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Non-limiting examples of leukemia diseases include, acute nonlymphocyticleukemia, chronic lymphocytic leukemia, acute granulocytic leukemia,chronic granulocytic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, a leukocythemic leukemia,basophilic leukemia, blast cell leukemia, bovine leukemia, chronicmyelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilicleukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia,stem cell leukemia, subleukemic leukemia, undifferentiated cellleukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblasticleukemia, histiocytic leukemia, stem cell leukemia, acute monocyticleukemia, leukopenic leukemia, lymphatic leukemia, lymphoblasticleukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoidleukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblastic leukemia, myelocytic leukemia, myeloid granulocyticleukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cellleukemia, plasmacytic leukemia, and promyelocytic leukemia.

In some embodiments, the methods and compositions provided herein relateto the treatment of a carcinoma. The term “carcinoma” refers to amalignant growth made up of epithelial cells tending to infiltrate thesurrounding tissues, and/or resist physiological and non-physiologicalcell death signals and gives rise to metastases. Non-limiting exemplarytypes of carcinomas include, acinar carcinoma, acinous carcinoma,adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,carcinoma of adrenal cortex, alveolar carcinoma, alveolar cellcarcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiennoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma,solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma,carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma,string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes,transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma,verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypernephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, and carcinomascroti.

In some embodiments, the methods and compositions provided herein relateto the treatment of a sarcoma. The term “sarcoma” generally refers to atumor which is made up of a substance like the embryonic connectivetissue and is generally composed of closely packed cells embedded in afibrillar, heterogeneous, or homogeneous substance. Sarcomas include,but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromalsarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giantcell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolarsoft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromasarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

Additional exemplary neoplasias that can be treated using the methodsand compositions described herein include Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,plasmacytoma, colorectal cancer, rectal cancer, and adrenal corticalcancer.

In some embodiments, the cancer treated is a melanoma. The term“melanoma” is taken to mean a tumor arising from the melanocytic systemof the skin and other organs. Non-limiting examples of melanomas areHarding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma,benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Particular categories of tumors that can be treated using methods andcompositions described herein include lymphoproliferative disorders,breast cancer, ovarian cancer, prostate cancer, cervical cancer,endometrial cancer, bone cancer, liver cancer, stomach cancer, coloncancer, pancreatic cancer, cancer of the thyroid, head and neck cancer,cancer of the central nervous system, cancer of the peripheral nervoussystem, skin cancer, kidney cancer, as well as metastases of all theabove. Particular types of tumors include hepatocellular carcinoma,hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma,thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma,invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonarysquamous cell carcinoma, basal cell carcinoma, adenocarcinoma (welldifferentiated, moderately differentiated, poorly differentiated orundifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma,hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testiculartumor, lung carcinoma including small cell, non-small and large celllung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma,colon carcinoma, rectal carcinoma, hematopoietic malignancies includingall types of leukemia and lymphoma including: acute myelogenousleukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia,multiple myeloma, myeloid lymphoma, Hodgkin' s lymphoma, non-Hodgkin' slymphoma, plasmacytoma, colorectal cancer, and rectal cancer.

Cancers treated in certain embodiments also include precancerouslesions, e.g., actinic keratosis (solar keratosis), moles (dysplasticnevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett'sesophagus, atrophic gastritis, dyskeratosis congenita, sideropenicdysphagia, lichen planus, oral submucous fibrosis, actinic (solar)elastosis and cervical dysplasia.

Cancers treated in some embodiments include non-cancerous or benigntumors, e.g., of endodermal, ectodermal or mesenchymal origin,including, but not limited to cholangioma, colonic polyp, adenoma,papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renaltubular adenoma, squamous cell papilloma, gastric polyp, hemangioma,osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma,rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.

Other Diseases and Disorders

In some embodiments, the methods and compositions described hereinrelate to the treatment of Nonalcoholic Fatty Liver Disease (NAFLD) andNonalcoholic Steatohepatitis (NASH).

In some embodiments, the methods and compositions described hereinrelate to the treatment of liver diseases. Such diseases include, butare not limited to, Alagille Syndrome, Alcohol-Related Liver Disease,Alpha-1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign LiverTumors, Biliary Atresia, Cirrhosis, Galactosemia, Gilbert Syndrome,Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, HepaticEncephalopathy, Intrahepatic Cholestasis of Pregnancy (ICP), LysosomalAcid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, NewbornJaundice, Non-Alcoholic Fatty Liver Disease, Primary Biliary Cholangitis(PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type IGlycogen Storage Disease, and Wilson Disease

The methods and compositions described herein may be used to treatneurodegenerative and neurological diseases. In certain embodiments, theneurodegenerative and/or neurological disease is Parkinson's disease,Alzheimer's disease, prion disease, Huntington's disease, motor neuronediseases (MND), spinocerebellar ataxia, spinal muscular atrophy,dystonia, idiopathicintracranial hypertension, epilepsy, nervous systemdisease, central nervous system disease, movement disorders, multiplesclerosis, encephalopathy , peripheral neuropathy or post-operativecognitive dysfunction.

Methods of Making Enhanced Bacteria

In certain aspects, provided herein are methods of making engineeredbacteria for the production of the EVs described herein. In someembodiments, the engineered bacteria are modified to enhance certaindesirable properties. For example, in some embodiments, the engineeredbacteria are modified to increase production of EVs by the bacteria. Insome embodiments, the engineered bacteria are modified to produce EVswith enhanced oral delivery (e.g., by improving acid resistance and/orresistance to bile acids), to enhance the immunomodulatory and/ortherapeutic effect of the EVs they produce (e.g., either alone or incombination with another therapeutic agent), to enhance immuneactivation by the EVs they produce and/or to improve bacterial and/or EVmanufacturing (e.g., higher oxygen tolerance, improved freeze-thawtolerance, shorter generation times). The engineered bacteria may beproduced using any technique known in the art, including but not limitedto site-directed mutagenesis, transposon mutagenesis, knock-outs,knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis,ultraviolet light mutagenesis, transformation (chemically or byelectroporation), phage transduction, directed evolution, CRISPR/Cas9,or any combination thereof.

In some embodiments of the methods provided herein, the bacterium ismodified by directed evolution. In some embodiments, the directedevolution comprises exposure of the bacterium to an environmentalcondition and selection of bacterium with improved survival and/orgrowth under the environmental condition. In some embodiments, themethod comprises a screen of mutagenized bacteria using an assay thatidentifies enhanced bacterium. In some embodiments, the method furthercomprises mutagenizing the bacteria (e.g., by exposure to chemicalmutagens and/or UV radiation) followed by an assay to detect bacteriahaving the desired phenotype (e.g., an in vivo assay, an ex vivo assay,or an in vitro assay).

In some embodiments, the bacterium provided herein are modified byexposure to a stress-inducing environment (e.g., an environment thatinduces envelope stress). In some embodiments, growth under such growthconditions increase production of EVs by the bacterium. For example, insome embodiments, the bacterium is grown in the presence ofsubinhibitory concentrations of an antibiotic described herein (e.g.,0.1-1 82 g/mL chloramphenicol, or 0.1-0.3 μg/mL gentamicin). In someembodiments, host antimicrobial peptides (e.g., lysozyme, defensins, andReg proteins) are used in place of or in combination with antibiotics.In some embodiments, bacterially-produced antimicrobial peptides (e.g.,bacteriocins and microcins) are used. In some embodiments, the stress istemperature stress (e.g., growth at 37-50° C.). In some embodiments, thestress is carbon limitation stress (e.g., growth in a media comprisinglimited carbon sources, such as media with carbon source restrictedbelow 1% (w/v)). In some embodiments, the stress is salt stress (e.g.,growth in a medium containing 0.5M NaCl). In some embodiments, thestress is UV stress (e.g., growth under a UV lamp, either throughout theentire cultivation period or only during a portion of the cultivationperiod). In some embodiments, the stress is reactive oxygen stress(e.g., growth in media containing subinhibitory concentrations ofhydrogen peroxide, such as 250-1,000 μM hydrogen peroxide). In someembodiments, a combination of the stresses disclosed herein are appliedto the bacterium.

EXAMPLES Example 1 Preparation and Purification of EVs from Bacteria.

Extracellular vesicles (EVs) are prepared from bacterial cultures usingmethods known to those skilled in the art (S. Bin Park, et al. PLoS ONE.6(3):e17629 (2011)).

For example, bacterial cultures are centrifuged at 11,000×g for 20-40min at 4° C. to pellet bacteria. Culture supernatants are then passedthrough a 0.22 μm filter to exclude intact bacterial cells. Filteredsupernatants are concentrated using methods that may include, but arenot limited to, ammonium sulfate precipitation, ultracentrifugation, orfiltration. Briefly, for ammonium sulfate precipitation, 1.5-3 Mammonium sulfate is added to filtered supernatant slowly, while stirringat 4° C. Precipitations are incubated at 4° C. for 8-48 hours and thencentrifuged at 11,000×g for 20-40 min at 4° C. The pellets containbacterial EVs and other debris. Briefly, using ultracentrifugation,filtered supernatants are centrifuged at 100,000-200,000×g for 1-16hours at 4° C. The pellet of this centrifugation contains bacterial EVsand other debris. Briefly, using a filtration technique, using an AmiconUltra spin filter or by tangential flow filtration, supernatants arefiltered so as to retain species of molecular weight >50 or 100 kDa.

Alternatively, EVs are obtained from bacterial cultures continuouslyduring growth, or at selected time points during growth, by connecting abioreactor to an alternating tangential flow (ATF) system (e.g., XCellATF from Repligen) according to manufacturer's instructions. The ATFsystem retains intact cells (>0.22 um) in the bioreactor, and allowssmaller components (e.g., EVs, free proteins) to pass through a filterfor collection. For example, the system may be configured so that the<0.22 um filtrate is then passed through a second filter of 100 kDa,allowing species such as EVs between 0.22 um and 100 kDa to becollected, and species smaller than 100 kDa to be pumped back into thebioreactor. Alternatively, the system may be configured to allow formedium in the bioreactor to be replenished and/or modified during growthof the culture. EVs collected by this method may be further purifiedand/or concentrated by ultracentrifugation or filtration as describedabove for filtered supernatants.

EVs obtained by methods described above may be further purified bygradient ultracentrifugation, using methods that may include, but arenot limited to, use of a sucrose gradient or Optiprep gradient. Briefly,using a sucrose gradient method, if ammonium sulfate precipitation orultracentrifugation were used to concentrate the filtered supernatants,pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. Iffiltration was used to concentrate the filtered supernatant, theconcentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0,using an Amicon Ultra column. Samples are applied to a 35-60%discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24hours at 4° C. Briefly, using an Optiprep gradient method, if ammoniumsulfate precipitation or ultracentrifugation were used to concentratethe filtered supernatants, pellets are resuspended in 35% Optiprep inPBS. If filtration was used to concentrate the filtered supernatant, theconcentrate is diluted using 60% Optiprep to a final concentration of35% Optiprep. Samples are applied to a 35-60% discontinuous sucrosegradient and centrifuged at 200,000×g for 3-24 hours at 4° C.

To confirm sterility and isolation of the EV preparations, EVs areserially diluted onto agar medium used for routine culture of thebacteria being tested, and incubated using routine conditions.Non-sterile preparations are passed through a 0.22 um filter to excludeintact cells. To further increase purity, isolated EVs may be DNase orproteinase K treated.

Alternatively, for preparation of EVs used for in vivo injections,purified EVs are processed as described previously (G. Norheim, et al.PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradientcentrifugation, bands containing EVs are resuspended to a finalconcentration of 50 μg/mL in a solution containing 3% sucrose or othersolution suitable for in vivo injection known to one skilled in the art.This solution may also contain adjuvant, for example aluminum hydroxideat a concentration of 0-0.5% (w/v).

To make samples compatible with further testing (e.g. to remove sucroseprior to TEM imaging or in vitro assays), samples are buffer exchangedinto PBS or 30 mM Tris, pH 8.0 using filtration (e.g. Amicon Ultracolumns), dialysis, or ultracentrifugation (200,000×g, >3 hours, 4° C.)and resuspension.

Example 2 Labeling Bacterial EVs

In order to track their biodistribution in vivo and to quantify andlocalize them in vitro in various preparations and in assays conductedwith mammalian cells, EVs are labeled as previously described (N. Kesty,et al. EMBO Journal. 23: 4538-4549 (2004)).

For example, purified EVs are incubated with fluorescein isothiocyanate(FITC) (Sigma-Aldrich, USA), Cy7, or any other fluorochrome suitable forflow cytometry, 1:1 for 1 hour at 25° C. The incubation step may beextended overnight at 4° C. To remove extra fluorochrome, EVs are theneither (1) pelleted by centrifugation at 200,000×g for 3 hr-overnight,washed, and resuspended in PBS or another appropriate buffer fordownstream applications; or (2) buffer exchanged into PBS or anotherappropriate buffer for downstream applications by dialysis or byfiltration (e.g., using an Amicon Ultra column).

Alternatively, EVs are obtained from bacteria cultured in mediumcontaining 0.8 mM 3-azido-D-alanine or HADA. EVs are resuspended orbuffer exchanged into PBS and a portion is further labeled with 10uMDibenzo-aza-cyclooctyne (DIBAC)-fluorescent dye in 1%BSA/PBS (dyesinclude CyS, TAMRA, Rhodamine-green, and Cy7) if grown with3-azido-D-alanine. Unincorporated dye is removed as described above, by(1) ultracentrifugation, washing, and resuspension; or (2) bufferexchange by dialysis or filtration.

Labeled EVs may also be generated from bacteria expressinggreen-fluorescent protein (GFP), or any other fluorescent protein. ForGram negative bacteria, periplasmic targeting sequences are appended tothe fluorescent proteins, so that they are appropriately localized to beinternalized by EVs as they form.

Quantum dots may be used to label EVs for non-invasive in vivo imagingstudies (K. Kikushima, et al. Scientific Reports. 3(1913) (2013)).Quantum dots are conjugated to an antibody confirmed to be present inthe EV membrane. Isolated EVs are incubated with quantum dot conjugates,and extra conjugates are removed as described above, by (1)ultracentrifugation, washing, and resuspension; or (2) buffer exchangeby dialysis or filtration.

Fluorescently labeled EVs are detected in in vitro and ex vivo samplesby confocal microscopy, nanoparticle tracking analysis, and/or flowcytometry. Additionally, fluorescently labeled EVs are detected in wholeanimals and/or dissected organs and tissues using an instrument such asthe IVIS spectrum CT (Perkin Elmer), as in H-I. Choi, et al.Experimental & Molecular Medicine. 49: e330 (2017).

Additionally, EVs may be radiolabeled as previously described (Z. Vargaet al., Cancer Biother Radiopharm. 2016 June;31(5):168-73).

For example, purified EVs are radiolabeled with the ⁹⁹mTc-tricarbonylcomplex [^(99m)Tc(CO)₃(H₂O)₃]⁺ using a commercial kit (Isolink®;Mallinckrodt Medical B.V.), according to the manufacturer'sinstructions.

Example 3 Transmission Electron Microscopy to Visualize BacterialProduction of EVs and Purified Bacterial EVs

Transmission electron microscopy (TEM) is used to visualize bacteria asthey produce EVs or purified bacterial EVs (S. Bin Park, et al. PLoSONE. 6(3):e17629 (2011). EVs are prepared from bacteria batch culture asdescribed in Example 1. EVs are mounted onto 300- or or 400-mesh-sizecarbon-coated copper grids (Electron Microscopy Sciences, USA) for 2 minand washed with deionized water. EVs are negatively stained using 2%(w/v) uranyl acetate for 20 sec-1 min. Copper grids are washed withsterile water and dried. Images are acquired using a transmissionelectron microscope with 100-120 kV acceleration voltage. Stained EVsappear between 20-250 nm in diameter and are electron dense. 10-50fields on each grid are screened.

Example 4 Profiling EV Composition and Content

EVs may be characterized by any one of various methods including, butnot limited to, NanoSight characterization, SDS-PAGE gelelectrophoresis, Western blot, ELISA, liquid chromatography-massspectrometry and mass spectrometry, dynamic light scattering, lipidlevels, total protein, lipid to protein ratios, nucleic acid analysisand zeta potential.

NanoSight Characterization of EVs

Nanoparticle tracking analysis (NTA) is used to characterize the sizedistribution of purified bacterial EVs. Purified EV preps are run on aNanoSight machine (Malvern Instruments) to assess EV size andconcentration.

SDS-PAGE Gel Electrophoresis

To identify the protein components of purified EVs (Example 1), samplesare run on a Bolt Bis-Tris Plus 4-12% gel (Thermo-Fisher Scientific)using standard techniques. Samples are boiled in lx SDS sample bufferfor 10 min, cooled to 4° C., and then centrifuged at 16,000×g for 1 min.Samples are then run on a SDS-PAGE gel and stained using one of severalstandard techniques (e.g., Silver staining, Coomassie Blue, Gel CodeBlue) for visualization of bands.

Western Blot Analysis

To identify and quantify specific protein components of purified EVs, EVproteins are separated by SDS-PAGE as described above and subjected toWestern blot analysis (Cvjetkovic et al., Sci. Rep. 6, 36338 (2016) andare quantified via ELISA.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and Mass Spectrometry(MS)

Proteins present in EVs are identified and quantified by MassSpectrometry techniques. Additionally, metabolic content is ascertainedusing liquid chromatography techniques combined with mass spectrometry.A variety of techniques exist to determine metabolomic content ofvarious samples and are known to one skilled in the art involvingsolvent extraction, chromatographic separation and a variety ofionization techniques coupled to mass determination (Roberts et al 2012Targeted Metabolomics. Curr Protoc Mol Biol. 30: 1-24; Dettmer et al2007, Mass spectrometry-based metabolomics. Mass Spectrom Rev.26(1):51-78). As a non-limiting example, a LC-MS system includes a 4000QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with 1100Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies).Media samples or other complex metabolic mixtures (˜10 μL) are extractedusing nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formicacid containing stable isotope-labeled internal standards (valine-d8,Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). Standardsmay be adjusted or modified depending on the metabolites of interest.The samples are centrifuged (10 min, 9,000g, 4° C.), and thesupernatants (10 μL) are submitted to LCMS by injecting the solutiononto the HILIC column (150×2.1 mm, 3 μm particle size). The column iseluted by flowing a 5% mobile phase [10 mM ammonium formate, 0.1% formicacid in water] for 1 min at a rate of 250 uL/min followed by a lineargradient over 10 min to a solution of 40% mobile phase [acetonitrilewith 0.1% formic acid]. The ion spray voltage is set to 4.5 kV and thesource temperature is 450° C.

The data are analyzed using commercially available software likeMultiquant 1.2 from AB SCIEX for mass spectrum peak integration. Peaksof interest should be manually curated and compared to standards toconfirm the identity of the peak. Quantitation with appropriatestandards is performed to determine the number of metabolites present inthe initial media, after bacterial conditioning and after tumor cellgrowth.

Dynamic light scattering (DLS)

DLS measurements, including the distribution of particles of differentsizes in different EV preps are taken using instruments such as theDynaPro NanoStar (Wyatt Technology) and the Zetasizer Nano ZS (MalvernInstruments).

Lipid Levels

Lipid levels are quantified using FM4-64 (Life Technologies), by methodssimilar to those described by A. J. McBroom etal. J Bacteriol188:5385-5392. and A. Frias, etal. Microb Ecol. 59:476-486 (2010).Samples are incubated with FM4-64 (3.3 μg/mL in PBS for 10 min at 37° C.in the dark). After excitation at 515 nm, emission at 635 nm is measuredusing a Spectramax M5 plate reader (Molecular Devices). Absoluteconcentrations are determined by comparison of unknown samples tostandards (such as palmitoyloleoylphosphatidylglycerol (POPG) vesicles)of known concentrations.

Total Protein

Protein levels are quantified by standard assays such as the Bradfordand BCA assays. The Bradford assays are run using Quick Start Bradfordlx Dye Reagent (Bio-Rad), according to manufacturer's protocols. BCAassays are run using the Pierce BCA Protein Assay Kit (Thermo-FisherScientific). Absolute concentrations are determined by comparison to astandard curve generated from BSA of known concentrations.

Lipid:Protein Ratios

Lipid:protein ratios are generated by dividing lipid concentrations byprotein concentrations. These provide a measure of the purity ofvesicles as compared to free protein in each preparation.

Nucleic acid analysis

Nucleic acids are extracted from EVs and quantified using a Qubitfluorometer. Size distribution is assessed using a BioAnalyzer and thematerial is sequenced.

Zeta Potential

The zeta potential of different preparations are measured usinginstruments such as the Zetasizer ZS (Malvern Instruments).

Example 5 Manipulating Bacteria Through Stress to Produce VariousAmounts of EVs and/or to Vary Content of EVs

Stress, and in particular envelope stress, has been shown to increaseproduction of EVs by some bacterial strains (I. MacDonald, M. Kuehn. JBacteriol 195(13): doi: 10/1128/JB.02267-12). In order to varyproduction of EVs by bacteria, bacteria are stressed using variousmethods.

Bacteria may be subjected to single stressors or stressors incombination. The effects of different stressors on different bacteria isdetermined empirically by varying the stress condition and determiningthe IC50 value (the conditions required to inhibit cell growth by 50%).EV purification, quantification, and characterization occurs as detailedin Examples 1-4. EV production is quantified (1) in complex samples ofbacteria and EVs by nanoparticle tracking analysis (NTA) or transmissionelectron microscopy (TEM); or (2) following EV purification by NTA,lipid quantification, or protein quantification. EV content is assessedfollowing purification by methods described above.

Antibiotic Stress

Bacteria are cultivated under standard growth conditions with theaddition of subinhibitory concentrations of antibiotics. This mayinclude 0.1-1 μg/mL chloramphenicol, or 0.1-0.3 μg/mL gentamicin, orsimilar concentrations of other antibiotics (e.g., ampicillin, polymyxinB). Host antimicrobial products such as lysozyme, defensins, and Regproteins may be used in place of antibiotics. Bacterially-producedantimicrobial peptides, including bacteriocins and microcins may also beused.

Temperature Stress

Bacteria are cultivated under standard growth conditions, but at higheror lower temperatures than are typical for their growth. Alternatively,bacteria are grown under standard conditions, and then subjected to coldshock or heat shock by incubation for a short period of time at low orhigh temperatures respectively. For example, bacteria grown at 37° C.are incubated for 1 hour at 4° C-18° C. for cold shock or 42° C-50° C.for heat shock.

Starvation and Nutrient Limitation

To induce nutritional stress, bacteria are cultivated under conditionswhere one or more nutrients are limited. Bacteria may be subjected tonutritional stress throughout growth or shifted from a rich medium to apoor medium. Some examples of media components that are limited arecarbon, nitrogen, iron, and sulfur. An example medium is M9 minimalmedium (Sigma-Aldrich), which contains low glucose as the sole carbonsource. Particularly for Prevotella spp., iron availability is varied byaltering the concentration of hemin in media and/or by varying the typeof porphyrin or other iron carrier present in the media, as cells grownin low hemin conditions were found to produce greater numbers of EVs (S.Stubbs et al. Letters in Applied Microbiology. 29:31-36 (1999). Mediacomponents are also manipulated by the addition of chelators such asEDTA and deferoxamine.

Saturation

Bacteria are grown to saturation and incubated past the saturation pointfor various periods of time. Alternatively, conditioned media is used tomimic saturating environments during exponential growth. Conditionedmedia is prepared by removing intact cells from saturated cultures bycentrifugation and filtration, as described in Example 1, andconditioned media may be further treated to concentrate or removespecific components.

Salt Stress

Bacteria are cultivated in or exposed for brief periods to mediumcontaining NaCl, bile salts, or other salts.

UV Stress

UV stress is achieved by cultivating bacteria under a UV lamp or byexposing bacteria to UV using an instrument such as a Stratalinker(Agilent). UV may be administered throughout the entire cultivationperiod, in short bursts, or for a single defined period followinggrowth.

Reactive Oxygen Stress

Bacteria are cultivated in the presence of subinhibitory concentrationsof hydrogen peroxide (250-1,000 μM) to induce stress in the form ofreactive oxygen species. Anaerobic bacteria are cultivated in or exposedto concentrations of oxygen that are toxic to them.

Detergent stress

Bacteria are cultivated in or exposed to detergent, such as sodiumdodecyl sulfate (SDS) or deoxycholate.

pH stress

Bacteria are cultivated in or exposed for limited times to media ofdifferent pH.

Example 6 Preparation of EV-free Bacteria

Bacterial samples containing minimal amounts of EVs are prepared. EVproduction is quantified (1) in complex samples of bacteria andextracellular components by NTA or TEM; or (2) following EV purificationfrom bacterial samples, by NTA, lipid quantification, or proteinquantification.

a. Centrifugation and washing: Bacterial cultures are centrifuged at11,000×g to separate intact cells from supernatant (including freeproteins and vesicles). The pellet is washed with buffer, such as PBS,and stored in a stable way (e.g., mixed with glycerol, flash frozen, andstored at −80° C.).

b. ATF: Bacteria and EVs are separated by connection of a bioreactor toan ATF system. EV-free bacteria are retained within the bioreactor, andmay be further separated from residual EVs by centrifugation andwashing, as described above.

c. Bacteria are grown under conditions that are found to limitproduction of EVs. Conditions that may be varied include those listedfor Example 5.

Example 7 In vitro Screening of EVs for Enhanced Activation of DendriticCells

The ability of Vibrio cholerae EVs to activate dendritic cellsindirectly through epithelial cells is one nonlimiting mechanism bywhich they stimulate an immune response in mammalian hosts (D.Chatterjee, K. Chadhuri. JBiol Chem. 288(6):4299-309. (2013)). As thisEV activity is likely shared with other bacteria that stimulatepro-inflammatory cascades in vivo, in vitro methods to assay DCactivation by bacterial EVs are disclosed herein. Briefly, PBMCs areisolated from heparinized venous blood from CMs by gradientcentrifugation using Lymphoprep (Nycomed, Oslo, Norway) or from mousespleens or bone marrow using the magnetic bead-based Human BloodDendritic cell isolation kit (Miltenyi Biotech, Cambridge, Mass.). Usinganti-human CD14 mAb, the monocytes are purified by Moflo and cultured incRPMI at a cell density of 5e5 cells/ml in a 96-well plate (Costar Corp)for 7 days at 37° C. For maturation of dendritic cells, the culture isstimulated with 0.2 ng/mL IL-4 and 1000 U/ml GM-CSF at 37° C. for oneweek. Alternatively, maturation is achieved through incubation withrecombinant GM-CSF alone for a week. Mouse DCs can be harvested directlyfrom spleens using bead enrichment or differentiated from haematopheoticstem cells. Briefly, bone marrow is obtained from the femurs of mice.Cells are recovered and red blood cells lysed. Stem cells are culturedin cell culture medium together with 20ng/m1 mouse GMCSF for 4 days.Additional medium containing 20ng/m1 mouse GM-CSF is added. On day 6 themedium and non-adherent cells are removed and replaced with fresh cellculture medium containing 20ng/m1 GMCSF. A final addition of cellculture medium with 20ng/m1 GM-CSF is added on day 7. On day 10non-adherent cells are harvested and seeded into cell culture platesovernight and stimulated as required. Dendritic cells are then treatedwith 25-75 ug/mL EVs for 24 hours with antibiotics. EV compositionstested may include a EVs from a single bacterial species or strain. EVcompositions tested may also include a mixture of EVs from bacterialgenera, species within a genus, or strains within a species. PBS and EVsfrom Lactobacillus are included as negative controls and LPS, anti-CD40antibodies, and EVs from Bifidobacterium spp. are used as positivecontrols. Following incubation, DCs are stained with anti CD11b, CD11c,CD103, CD8a, CD40, CD80, CD83, CD86, MHCI and MHCII, and analyzed byflow cytometry. DCs that are significantly increased in CD40, CD80,CD83, and CD86 as compared to negative controls are considered to beactivated by the associated bacterial EV composition. These experimentsare repeated three times at minimum.

To screen for the ability of EV-activated epithelial cells to stimulateDCs, the above protocol is followed with the addition of a 24-hourepithelial cell EV co-culture prior to incubation with DCs. Epithelialcells are washed after incubation with EVs and are then co-cultured withDCs in an absence of EVs for 24 hours before being processed as above.Epithelial cell lines may include Int407, HEL293, HT29, T84 and CACO2.

As an additional measure of DC activation, 100 μl of culture supernatantis removed from wells following 24 hour incubation of DCs with EVs orEV-treated epithelial cells and is analyzed for secreted cytokines,chemokines, and growth factors using the multiplexed Luminex Magpix. Kit(EMD Millipore, Darmstadt, Germany). Briefly, the wells are pre-wet withbuffer, and 25 μl of 1× antibody-coated magnetic beads are added and 2×200 μl of wash buffer are performed in every well using the magnet. 50μl of Incubation buffer, 50 μl of diluent and 50 μl of samples are addedand mixed via shaking for 2 hrs at room temperature in the dark. Thebeads are then washed twice with 200 μl wash buffer. 100 μl of 1×biotinylated detector antibody is added and the suspension is incubatedfor 1 hr with shaking in the dark. Two, 200 μl washes are then performedwith wash buffer. 100 μl of 1× SAV-RPE reagent is added to each well andis incubated for 30 min at RT in the dark. Three 200 μl washes areperformed and 125 μl of wash buffer is added with 2-3 min shakingoccurs. The wells are then submitted for analysis in the Luminex xMAPsystem.

Standards allow for careful quantitation of the cytokines includingGM-CSF, IFN-g, IFN-a, IFN-B, IL-la, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8,IL-10, IL-13, IL-12 (p40/p70), IL-17A, IL-17F, IL-21, IL-22 IL-23,IL-25, IP-10, KC, MCP-1, MIG, MIP1a, TNFa, and VEGF. These cytokines areassessed in samples of both mouse and human origin. Increases in thesecytokines in the bacterial treated samples indicate enhanced productionof proteins and cytokines from the host. Other variations on this assayexamining specific cell types ability to release cytokines are assessedby acquiring these cells through sorting methods and are recognized byone of ordinary skill in the art. Furthermore, cytokine mRNA is alsoassessed to address cytokine release in response to a EV composition.These changes in the cells of the host stimulate an immune responsesimilarly to in vivo response in a cancer microenvironment.

This DC stimulation protocol may be repeated using combinations ofpurified EVs and live bacterial strains to maximize immune stimulationpotential.

Example 8 In vitro Screening of EVs for Enhanced Activation of CD8+ TCell Killing When Incubated with Tumor Cells

In vitro methods for screening for EVs that can activate CD8+ T cellkilling of tumor cells are described. Briefly, DCs are isolated fromhuman PBMCs or mouse spleens and incubated with single-strain EVs,mixtures of EVs, and appropriate controls as described in Example 12. Inaddition, CD8+ T cells are obtained from human PBMCs or mouse spleensusing the magnetic bead-based Mouse CD8a+ T Cell Isolation Kit and themagnetic bead-based Human CD8+ T Cell Isolation Kit (both from MiltenyiBiotech, Cambridge, Mass.). After the 24 hour incubation of DCs withEVs, or DCs with EV-stimulated epithelial cells (detailed in Example12), EVs are removed from cells with PBS washes, 100 μl of fresh mediawith antibiotics is added to each well, and 200,000 T cells are added toeach experimental well in the 96-well plate. Anti-CD3 antibody is addedat a final concentration of 2ug/ml. Co-cultures are then allowed toincubate at 37° C. for 96 hours under normal oxygen conditions.

72 hours into the coculture incubation, 50,000 tumor cells/well areplated per well in new 96-well plates. Mouse tumor cell lines usedinclude B16.F10, SIY+B16.F10, and others. Human tumor cell lines areHLA-matched to donor, and can include PANC-1, UNKPC960/961, UNKC, andHELA cell lines. After completion of the 96 hour co-culture, 100 μl ofthe CD8+ T cell and DC mixture is transferred to wells containing tumorcells. Plates are incubated for 24 hours at 37° C. under normal oxygenconditions. Staurospaurine is used as negative control to account forcell death.

Following this incubation, flow cytometry is used to measure tumor celldeath and characterize immune cell phenotype. Briefly, tumor cells arestained with viability dye. FACS analysis is used to gate specificallyon tumor cells and measure the percentage of dead (killed) tumor cells.Data are also displayed as the absolute number of dead tumor cells perwell. Cytotoxic CD8+ T cell phenotype may be characterized by thefollowing methods: a) concentration of supernatant granzyme B, IFNy andTNFa in the culture supernatant as described below, b) CD8+ T cellsurface expression of activation markers such as DC69, CD25, CD154,PD-1, gamma/delta TCR, Foxp3, T-bet, granzyme B, c) intracellularcytokine staining of IFNy, granzyme B, TNFa in CD8+ T cells. CD4+ T cellphenotype may also be assessed by intracellular cytokine staining inaddition to supernatant cytokine concentration including INFy, TNFa,IL-12, IL-4, IL-5, IL-17, IL-10, chemokines etc.

As an additional measure of CD8+ T cell activation, 100 μl of culturesupernatant is removed from wells following the 96 hour incubation of Tcells with DCs and is analyzed for secreted cytokines, chemokines, andgrowth factors using the multiplexed Luminex Magpix. Kit (EMD Millipore,Darmstadt, Germany). Briefly, the wells are pre-wet with buffer, and 25μl of 1× antibody-coated magnetic beads are added and 2× 200 μl of washbuffer are performed in every well using the magnet. 50 μl of Incubationbuffer, 50 μl of diluent and 50 μl of samples are added and mixed viashaking for 2hrs at room temperature in the dark. The beads are thenwashed twice with 200 μl wash buffer. 100 μl of 1× biotinylated detectorantibody is added and the suspension is incubated for 1 hr with shakingin the dark. Two, 200 μl washes are then performed with wash buffer. 100μl of 1× SAV-RPE reagent is added to each well and is incubated for 30min at RT in the dark. Three 200 μl washes are performed and 125 μl ofwash buffer is added with 2-3 min shaking occurs. The wells are thensubmitted for analysis in the Luminex xMAP system.

Standards allow for careful quantitation of the cytokines includingGM-CSF, IFN-g, IFN-a, IFN-B IL-la, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8,IL-10, IL-13, IL-12 (p40/p70), IL-17, IL-23, IP-10, KC, MCP-1, MIG,MIP1a, TNFa, and VEGF. These cytokines are assessed in samples of bothmouse and human origin. Increases in these cytokines in the bacterialtreated samples indicate enhanced production of proteins and cytokinesfrom the host. Other variations on this assay examining specific celltypes ability to release cytokines are assessed by acquiring these cellsthrough sorting methods and are recognized by one of ordinary skill inthe art. Furthermore, cytokine mRNA is also assessed to address cytokinerelease in response to an EV composition. These changes in the cells ofthe host stimulate an immune response similarly to in vivo response in acancer microenvironment.

This CD8+ T cell stimulation protocol may be repeated using combinationsof purified EVs and live bacterial strains to maximize immunestimulation potential.

Example 9 In vitro Screening of EVs for Enhanced Tumor Cell Killing byPBMCs

Methods to screen EVs for the ability to stimulate PBMCs, which in turnactivate CD8+ T cells to kill tumor cells are included. PBMCs areisolated from heparinized venous blood from CMs by ficoll-paque gradientcentrifugation for mouse or human blood, or with Lympholyte CellSeparation Media (Cedarlane Labs, Ontario, Canada) from mouse blood.PBMCs are incubated with single-strain EVs, mixtures of EVs, andappropriate controls as described in Example 12. In addition, CD8+ Tcells are obtained from human PBMCs or mouse spleens as in Example 12.After the 24 hour incubation of PBMCs with EVs, EVs are removed fromcells with PBS washes, 100 μl of fresh media with antibiotics is addedto each well, and 200,000 T cells are added to each experimental well inthe 96-well plate. Anti-CD3 antibody is added at a final concentrationof 2ug/ml. Co-cultures are then allowed to incubate at 37° C. for 96hours under normal oxygen conditions.

72 hours into the coculture incubation, 50,000 tumor cells/well areplated per well in new 96-well plates. Mouse tumor cell lines usedinclude B16.F10, SIY+B16.F10, and others. Human tumor cell lines areHLA-matched to donor, and can include PANC-1, UNKPC960/961, UNKC, andHELA cell lines. After completion of the 96 hour co-culture, 100 μl ofthe CD8+ T cell and PBMC mixture is transferred to wells containingtumor cells. Plates are incubated for 24 hours at 37° C. under normaloxygen conditions. Staurospaurine is used as negative control to accountfor cell death.

Following this incubation, flow cytometry is used to measure tumor celldeath and characterize immune cell phenotype. Briefly, tumor cells arestained with viability dye. FACS analysis is used to gate specificallyon tumor cells and measure the percentage of dead (killed) tumor cells.Data are also displayed as the absolute number of dead tumor cells perwell. Cytotoxic CD8+ T cell phenotype may be characterized by thefollowing methods: a) concentration of supernatant granzyme B, IFNy andTNFa in the culture supernatant as described below, b) CD8+ T cellsurface expression of activation markers such as DC69, CD25, CD154,PD-1, gamma/delta TCR, Foxp3, T-bet, granzyme B, c) intracellularcytokine staining of IFNy, granzyme B, TNFa in CD8+ T cells. CD4+T cellphenotype may also be assessed by intracellular cytokine staining inaddition to supernatant cytokine concentration including INFy, TNFa,IL-12, IL-4, IL-5, IL-17, IL-10, chemokines etc.

As an additional measure of CD8+ T cell activation, 100 μl A of culturesupernatant is removed from wells following the 96 hour incubation of Tcells with DCs and is analyzed for secreted cytokines, chemokines, andgrowth factors using the multiplexed Luminex Magpix. Kit (EMD Millipore,Darmstadt, Germany). Briefly, the wells are pre-wet with buffer, and 25μl A of lx antibody-coated magnetic beads are added and 2× 200 μl ofwash buffer are performed in every well using the magnet. 50 μl A ofIncubation buffer, 50 μl of diluent and 50 μl of samples are added andmixed via shaking for 2hrs at room temperature in the dark. The beadsare then washed twice with 200 μl wash buffer. 100 μl of 1× biotinylateddetector antibody is added and the suspension is incubated for 1 hr withshaking in the dark. Two, 200 μl washes are then performed with washbuffer. 100 μl of 1× SAV-RPE reagent is added to each well and isincubated for 30 min at RT in the dark. Three 200 μl washes areperformed and 125 μl of wash buffer is added with 2-3 min shakingoccurs. The wells are then submitted for analysis in the Luminex xMAPsystem.

Standards allow for careful quantitation of the cytokines includingGM-CSF, IFN-g, IFN-a, IFN-B IL-la, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8,IL-10, IL-13, IL-12 (p40/p70), IL-17, IL-23, IP-10, KC, MCP-1, MIG,MIP1a, TNFa, and VEGF. These cytokines are assessed in samples of bothmouse and human origin. Increases in these cytokines in the bacterialtreated samples indicate enhanced production of proteins and cytokinesfrom the host. Other variations on this assay examining specific celltypes ability to release cytokines are assessed by acquiring these cellsthrough sorting methods and are recognized by one of ordinary skill inthe art. Furthermore, cytokine mRNA is also assessed to address cytokinerelease in response to an EV composition. These changes in the cells ofthe host stimulate an immune response similarly to in vivo response in acancer microenvironment.

This PBMC stimulation protocol may be repeated using combinations ofpurified EVs and live bacterial strains to maximize immune stimulationpotential.

Example 10 In vitro Detection of EVs in Antigen-Presenting Cells

Dendritic cells in the lamina propria constantly sample live bacteria,dead bacteria, and microbial products in the gut lumen by extendingtheir dendrites across the gut epithelium, which is one way that EVsproduced by bacteria in the intestinal lumen may directly stimulatedendritic cells. The following methods represent a way to assess thedifferential uptake of EVs by antigen-presenting cells. Optionally,these methods may be applied to assess immunomodulatory behavior of EVsadministered to a patient.

Dendritic cells (DCs) are isolated from human or mouse bone marrow,blood, or spleens according to standard methods or kit protocols (e.g.,Inaba K, Swiggard W J, Steinman R M, Romani N, Schuler G, 2001.Isolation of dendritic cells. Current Protocols in Immunology. Chapter3:Unit3.7) and as discussed in Example 12.

To evaluate EV entrance into and/or presence in DCs, 250,000 DCs areseeded on a round cover slip in complete RPMI-1640 medium and are thenincubated with EVs from single bacterial strains or combinations EVs ata multiplicity of infection (MOI) between 1:1 and 1:10. Purified EVshave been labeled with fluorochromes or fluorescent proteins asdescribed in Example 2. After 1 hour of incubation, the cells are washedtwice with ice-cold PBS, detached from the plate using trypsin. Cellsare either allowed to remain intact or are lysed. Samples are thenprocessed for flow cytometry. Total internalized EVs are quantified fromlysed samples, and percentage of cells that uptake EVs is measured bycounting fluorescent cells. The methods described above may also beperformed in substantially the same manner using macrophages orepithelial cell lines (obtained from the ATCC) in place of DCs.

Example 11 In vitro Screening of EVs with an Enhanced Ability toActivate NK Cell Killing When Incubated with Target Cells

To demonstrate the ability of the selected EV compositions to elicitpotent NK cell cytotoxicity to tumor cells when incubated with the tumorcells, the following in vitro assay is used. Briefly, mononuclear cellsfrom heparinized blood are obtained from healthy human donors.Optionally, an expansion step to increase the numbers of NK cells isperformed as previously described (e.g. see Somanschi et al 2011 J VisExp.). They are adjusted to a concentration of 1e6 cells/ml in RPMI-1640medium containing 5% human serum. The PMNC cells are then labeled withappropriate antibodies and NK cells are isolated through FACS asCD3-/CD56+ cells and are ready for the subsequent cytotoxicity assay.Alternatively, NK cells are isolated using the autoMACs instrument andNK cell isolation kit following manufacturer's instructions (MiltenylBiotec).

NK cells are counted, and plated in a 96 well format with 5000 cells perwell, and incubated with single-strain EVs, EVs from mixtures ofbacterial strains, and appropriate controls as described in Example 12.As an additional negative control, this assay is run with EVs fromFusobacterium nucleatum. F. nucleatum is known to be inhibitory to NKcell activity (see e.g. Gur et al 2005 Immunity 42:1-12). After 5-24hours incubation of NK cells with EVs, EVs are removed from cells withPBS washes, NK cells are resuspended in 10 fresh media with antibiotics,and are added to 96-well plates containing 50,000 target tumorcells/well. Mouse tumor cell lines used include B16.F10, SIY+B16.F10,and others. Human tumor cell lines are HLA-matched to donor, and caninclude PANC-1, UNKPC960/961, UNKC, and HELA cell lines. Plates areincubated for 24 hours at 37° C. under normal oxygen conditions.Staurospaurine is used as negative control to account for cell death.

Following this incubation, flow cytometry is used to measure tumor celldeath. Briefly, tumor cells are stained with viability dye. FACSanalysis is used to gate specifically on tumor cells and measure thepercentage of dead (killed) tumor cells. Data are also displayed as theabsolute number of dead tumor cells per well.

This NK cell stimulation protocol may be repeated using combinations ofpurified EVs and live bacterial strains to maximize immune stimulationpotential.

Example 12 Using in Vitro Immune Activation Assays to Predict In vivoCancer Immunotherapy Efficacy of EV Compositions

In vitro immune activation assays identify EVs that are able tostimulate dendritic cells, which in turn activate CD8+ T cell killing.Work by A. Sivan, et al, Science 350(6264): 1084-1089 (2015) hassuggested that enhanced killing of tumor cells by CD8+ T cells inresponse to oral ingestion of Bifidobacterium spp. is an effectivecancer immunotherapy in mice. Therefore, the in vitro assays describedabove are used as a predictive, fast screen of a large number ofcandidate EVs for potential immunotherapy activity. EVs that displayenhanced stimulation of dendritic cells, enhanced stimulation of CD8+ Tcell killing, enhanced stimulation of PBMC killing, and/or enhancedstimulation of NK cell killing, are preferentially chosen for in vivocancer immunotherapy efficacy studies.

Example 13 Determining the Biodistribution of EVs When Delivered Orallyto Mice

Wild-type mice (e.g., C57BL/6 or BALB/c) are orally inoculated with theEV composition of interest to determine the in vivo biodistibutionprofile of purified EVs (Example 1). EVs are labeled as in Example 2 toaide in downstream analyses.

Mice can receive a single dose of the EV (25-100 μg) or several dosesover a defined time course (25-100 μg). Mice are housed under specificpathogen-free conditions following approved protocols. Alternatively,mice may be bred and maintained under sterile, Germ-free conditions.Blood and stool samples can be taken at appropriate time points.

The mice are humanely sacrificed at various time points (i.e., hours todays) post inoculation with the EV compositions and a full necropsyunder sterile conditions is performed. Following standard protocols,lymph nodes, adrenal glands, liver, colon, small intestine, cecum,stomach, spleen, kidneys, bladder, pancreas, heart, skin, lungs, brain,and other tissue of interest are harvested and are used directly or snapfrozen for further testing. The tissue samples are dissected andhomogenized to prepare single-cell suspensions following standardprotocols known to one skilled in the art. The number of EVs present inthe sample is then quantified through flow cytometry (Example 17).Quantification may also proceed with use of fluorescence microscopyafter appropriate processing of whole mouse tissue (Vankelecom H.,Fixation and paraffin-embedding of mouse tissues for GFP visualization,Cold Spring Harb. Protoc., 2009). Alternatively, the animals may beanalyzed using live-imaging according to the EV labeling technique.

Example 14 Administering EV Compositions with Enhanced Immune ActivationIn vitro to Treat Syngeneic Mouse Tumor Models

A mouse model of cancer is generated by subcutaneously injecting a tumorcell line or patient derived tumor sample and allowing it to engraftinto C57BL/6, female mice at ages 6-8 weeks old. The methods providedherein are replicated using several tumor cell lines including: B16-F10or B16-F10-SIY cells as an orthotopic model of melanoma, Panc02 cells asan orthotopic model of pancreatic cancer, injected at a concentration of1×10⁶ cells into the right flank (Maletzki et al 2008. Gut 57:483-491),LLC1 cells as an orthotopic model of lung cancer, CT-26 as an orthotopicmodel of colorectal cancer, and RM-1 as an orthotopic model of prostatecancer. As an example, methods for the B16-F10 model are provided indepth herein.

A syngeneic mouse model of spontaneous melanoma with a very highmetastatic frequency is used to test the ability of bacteria to reducetumor growth and the spread of metastases. The EVs chosen for this assayare compositions that display enhanced activation of immune cell subsetsand stimulate enhanced killing of tumor cells in vitro (Examples 12-16).The mouse melanoma cell line B16-F10 is obtained from ATCC. The cellsare cultured in vitro as a monolayer in RPMI medium, supplemented with10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycinat 37° C. in an atmosphere of 5% CO2 in air. The exponentially growingtumor cells are harvested by trypsinization, washed three times withcold 1× PBS, and a suspension of 5E6 cells/ml is prepared foradministration. Female C57BL/6 mice are used for this experiment. Themice are 6-8 weeks old and weigh approximately 16-20 g. For tumordevelopment, each mouse is injected SC into the flank with 100 μl of theB16-F10 cell suspension. The mice are anesthetized by ketamine andxylazine prior to the cell transplantation. The animals used in theexperiment may be started on an antibiotic treatment via instillation ofa cocktail of kanamycin (0.4 mg/ml), gentamicin, (0.035 mg/ml), colistin(850 U/ml), metronidazole (0.215 mg/ml) and vancomycin (0.045 mg/ml) inthe drinking water from day 2 to 5 and an intraperitoneal injection ofclindamycin (10 mg/kg) on day 7 after tumor injection.

The size of the primary flank tumor is measured with a caliper every 2-3days and the tumor volume is calculated using the following formula:tumor volume=the tumor width2×tumor length×0.5. After the primary tumorreaches approximately 100 mm3, the animals are sorted into severalgroups based on their body weight. The mice are then randomly taken fromeach group and assigned to a treatment group. EV compositions areprepared as described in Example 1. The mice are orally inoculated bygavage with either 25-100 μg EV to be tested, 25-100 μg EV fromLactobacillus (negative control), PBS, or 25-100 μg EV fromBifidobacterium spp. (positive control). Mice are orally gavaged withthe same amount of EVs daily, weekly, bi-weekly, monthly, bi-monthly, oron any other dosing schedule throughout the treatment period. Mice areIV injected in the tail vein or directly injected into the tumor. Micecan be injected with 10 ng-1 ug of EVs, bacteria and EVs or inactivatedbacteria and EVs. Mice can be injected weekly or once a month. Mice mayalso receive combinations of purified EVs and live bacteria to maximizetumor-killing potential. All mice are housed under specificpathogen-free conditions following approved protocols. Tumor size, mouseweight, and body temperature are monitored every 3-4 days and the miceare humanely sacrificed 6 weeks after the B16-F10 mouse melanoma cellinjection or when the volume of the primary tumor reaches 1000 mm3.Blood draws are taken weekly and a full necropsy under sterileconditions is performed at the termination of the protocol.

Cancer cells can be easily visualized in the mouse B16-F10 melanomamodel due to their melanin production. Following standard protocols,tissue samples from lymph nodes and organs from the neck and chestregion are collected and the presence of micro- and macro-metastases isanalyzed using the following classification rule. An organ is classifiedas positive for metastasis if at least two micro-metastatic and onemacro-metastatic lesion per lymph node or organ are found.Micro-metastases are detected by staining the paraffin-embedded lymphoidtissue sections with hematoxylin-eosin following standard protocolsknown to one skilled in the art. The total number of metastases iscorrelated to the volume of the primary tumor and it is found that thetumor volume correlates significantly with tumor growth time and thenumber of macro- and micro-metastases in lymph nodes and visceral organsand also with the sum of all observed metastases. Twenty-five differentmetastatic sites are identified as previously described (Bobek V., etal., Syngeneic lymph-node-targeting model of green fluorescentprotein-expressing Lewis lung carcinoma, Clin. Exp. Metastasis,2004;21(8):705-8).

The tumor tissue samples are further analyzed for tumor infiltratinglymphocytes. The CD8+ cytotoxic T cells can be isolated by FACS (seeExample 17) and can then be further analyzed using customized p/MHCclass I microarrays to reveal their antigen specificity (see e.g.Deviren G., et al., Detection of antigen-specific T cells on p/MHCmicroarrays, J. Mol. Recognit., 2007 Jan-Feb;20(1):32-8). CD4+T cellscan be analyzed using customized p/MHC class II microarrays.

The same experiment is also performed with a mouse model of multiplepulmonary melanoma metastases. The mouse melanoma cell line B16-BL6 isobtained from ATCC and the cells are cultured in vitro as describedabove. Female C57BL/6 mice are used for this experiment. The mice are6-8 weeks old and weigh approximately 16-20 g. For tumor development,each mouse is injected into the tail vein with 100 μlof a 2E6 cells/mlsuspension of B16-BL6 cells. The tumor cells that engraft upon IVinjection end up in the lungs.

The mice are humanely killed after 9 days. The lungs are weighed andanalyzed for the presence of pulmonary nodules on the lung surface. Theextracted lungs are bleached with Fekete's solution, which does notbleach the tumor nodules because of the melanin in the B16 cells thougha small fraction of the nodules is amelanotic (i.e. white). The numberof tumor nodules is carefully counted to determine the tumor burden inthe mice. Typically, 200-250 pulmonary nodules are found on the lungs ofthe control group mice (i.e. PBS gavage).

The percentage tumor burden is calculated for the three treatmentgroups. This measure is defined as the mean number of pulmonary noduleson the lung surfaces of mice that belong to a treatment group divided bythe mean number of pulmonary nodules on the lung surfaces of the controlgroup mice.

Determining metabolic content with H-NMR1

Biological triplicates of media and spent media samples after bacterialconditioning and after growth of the tumor are deproteinized usingSartorius Centrisart I filters (cutoff 10 kDa). Before use, the filteris washed twice by centrifugation of water to remove glycerol and asmall volume (20 μl) of 20.2 mMtrimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP, sodium salt) inD2O is added to 700 μl of the ultrafiltrate, providing a chemical shiftreference (0.00 ppm) and a deuterium lock signal. 650 μl of the sampleis placed in a 5 mm NMR tube. Single pulse 1H-NMR spectra (500 MHz) areobtained on a Bruker DMX-500 spectrometer or comparable instrument asdescribed previously (by Engelke et al. 2006 NMR spectroscopic studieson the late onset form of 3-methylutaconic aciduria type I and otherdefects in leucine metabolism. NMR Biomed. 19: 271-278). Phase andbaseline are corrected manually. All spectra are scaled to TSP andmetabolite signals are fitted semi-automatically with a Lorentzian lineshape. Metabolite concentrations in the spent media are calculatedrelative to the known concentration in the standard medium andcorrespondingly expressed in units of mM. The concentration of aparticular metabolite was calculated by the area of the correspondingpeak to the area of the valine doublet at 1.04 ppm or an appropriatestandard.

Determining metabolic content with LCMS

Metabolic content of a sample is ascertained using liquid chromatographytechniques combined with mass spectrometry. A variety of techniquesexist to determine metabolomic content of various samples and are knownto one skilled in the art involving solvent extraction, chromatographicseparation and a variety of ionization techniques coupled to massdetermination (Roberts et al 2012 Targeted Metabolomics. Curr Protoc MolBiol. 30: 1-24; Dettmer et al 2007, Mass spectrometry-basedmetabolomics. Mass Spectrom Rev. 26(1):51-78). As a non-limitingexample, a LC-MS system includes a 4000 QTRAP triple quadrupole massspectrometer (AB SCIEX) combined with 1100 Series pump (Agilent) and anHTS PAL autosampler (Leap Technologies). Media samples or other complexmetabolic mixtures (˜10 μL) are extracted using nine volumes of74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containingstable isotope-labeled internal standards (valine-d8, Isotec; andphenylalanine-d8, Cambridge Isotope Laboratories). Standards may beadjusted or modified depending on the metabolites of interest. Thesamples are centrifuged (10 min, 9,000g, 4 ° C.), and the supernatants(10 μL) are submitted to LCMS by injecting the solution onto the HILICcolumn (150×2.1 mm, 3 μm particle size). The column is eluted by flowinga 5% mobile phase [10mM ammonium formate, 0.1% formic acid in water] for1 min at a rate of 250 μL/min followed by a linear gradient over 10 minto a solution of 40% mobile phase [acetonitrile with 0.1% formic acid].The ion spray voltage is set to 4.5 kV and the source temperature is 450° C.

The data are analyzed using commercially available software such asMultiquant 1.2 from AB SCIEX for mass spectrum peak integration. Peaksof interest are manually curated and compared to standards to confirmthe identity of the peak. Quantitation with appropriate standards isperformed to determine the amount of metabolites present in the initialmedia, after bacterial conditioning and after tumor cell growth.

The tumor biopsies and blood samples are submitted for metabolicanalysis via LCMS techniques described herein. Differential levels ofamino acids, sugars, lactate, among other metabolites, between testgroups demonstrate the ability of the microbial composition to disruptthe tumor metabolic state.

RNA Seq to Determine Mechanism of Action

Dendritic cells are purified from tumors, Peyers patches, and mesentericlymph nodes as described in Example 12. RNAseq analysis is carried outand analyzed according to standard techniques known to one skilled inthe art (Z. Hou. Scientific Reports. 5(9570):doi:10.1038/srep09570(2015)). In the analysis, specific attention is placed on innateinflammatory pathway genes including TLRs, CLRs, NLRs, and STING,cytokines, chemokines, antigen processing and presentation pathways,cross presentation, and T cell co-stimulation.

Example 15 Administering EVs with Enhanced Immune Activation In vitro toTreat Syngeneic Mouse Tumor Models in Combination with PD-1 or PD-L1Inhibition

To determine the efficacy of EVs in syngeneic tumor mouse models,colorectal cancer (CT-26) was used. Briefly, CT-26 (CAT# CRL-2638) tumorcells were cultured in vitro as a monolayer in RPMI-1640 or DMEMsupplemented with 10% heat-inactivated fetal bovine serum at 37° C. inan atmosphere of 5% CO2 in air. The exponentially-growing cells wereharvested and counted prior to tumor inoculation. 6-8 week old femaleBALB/c mice were used for this experiment. For tumor development, eachmouse was injected subcutaneously in one or both rear flanks with 5×10⁵CT-26 tumor cells in 0.1 ml of 1× PBS. Some mice may receive antibioticpre-treatment. Tumor size and mouse weight were monitored at leastthrice weekly on nonconsecutive days.

EVs were tested for their efficacy in the mouse tumor model, eitheralone or in combination with whole bacterial cells and with or withoutanti-PD-1 or anti-PD-L1. EVs, bacterial cells, and/or anti-PD-1 oranti-PD-L1 were administered at varied time points and at varied doses.For example, on day 10 after tumor injection, or after the tumor volumereaches 100mm³, the mice were treated with EVs alone or in combinationwith anti-PD-1 or anti-PD-L1. Dosing amount was administered accordingto table 3. The Blautia massiliensis group showed tumor growthinhibition comparable to that seen in the anti-PD-1 group (FIGS. 1 and2).

TABLE 3 Dosing amount. Group Treatment Dose, route, schedule 1 (n = 10)IV Vehicle (PBS) N/A, IV, Q3Dx4 2 (n = 10) Anti-PD-1 200 ug, IP, Q4Dx3 3(n = 10) EV (IV) Blautia massiliensis 5 ug, IV, Q3Dx4

For example, some mice are intravenously injected with EVs at 15, 20, or15 ug/mouse. Other mice may receive 25, 50, or 100 mgs of EVs per mouse.While some mice receive EVs through i.v. injection, other mice mayreceive EVs through intraperitoneal (i.p.) injection, subcutaneous(s.c.) injection, nasal route administration, oral gavage, or othermeans of administration. Some mice may receive EVs every day (e.g.starting on day 1), while others may receive EVs at alternativeintervals (e.g. every other day, or once every three days). Additionalgroups of mice may receive some ratio of bacterial cells to EVs. Thebacterial cells may be live, dead, or weakened. The bacterial cells maybe harvested fresh (or frozen) and administered, or they may beirradiated or heat-killed prior to administration. For example, somegroups of mice may receive between 1×10⁴ and 5×10⁹ bacterial cells in anadministration separate from, or comingled with, the EV administration.As with the EVs, bacterial cell administration may be varied by route ofadministration, dose, and schedule. This can include oral gavage, i.v.injection, i.p. injection, or nasal route injection. Some groups of miceare also injected with effective doses of checkpoint inhibitor. Forexample, mice receive 100 μg anti-PD-L1 mAB (clone 10f.9g2, BioXCell) oranother anti-PD-1 or anti-PD-L1 mAB in 100 μl PBS, and some mice receivevehicle and/or other appropriate control (e.g. control antibody). Miceare injected with mABs 3, 6, and 9 days after the initial injection. Toassess whether checkpoint inhibition and EV immunotherapy have anadditive, anti-tumor effect, control mice receiving anti-PD-1 oranti-PD-L1 mABs are included to the standard control panel. Primary(tumor size) and secondary (tumor infiltrating lymphocytes and cytokineanalysis) endpoints are assessed, and some groups of mice arerechallenged with a subsequent tumor cell inoculation to assess theeffect of treatment on memory response.

Example 16 EVs in a Mouse Model of Experimental AutoimmuneEncephalomyelitis (EAE)

EAE is a well-studied animal model of multiple sclerosis, as reviewed byConstantinescu et al. (Experimental autoimmune encephalomyelitis (EAE)as a model for multiple sclerosis (MS). Br J Pharmacol. 2011 October;164(4): 1079-1106). It can be induced in a variety of mouse and ratstrains using different myelin-associated peptides, by the adoptivetransfer of activated encephalitogenic T cells, or the use of TCRtransgenic mice susceptible to EAE, as discussed in Mangalam et al. (Twodiscreet subsets of CD8+ T cells modulate PLP₉₁₋₁₁₀ induced experimentalautoimmune encephalomyelitis in HLA-DR3 transgenic mice. J Autoimmun.2012 June; 38(4): 344-353).

EVs are tested for their efficacy in the rodent model of EAE, eitheralone or in combination with whole bacterial cells, with or without theaddition of other anti-inflammatory treatments. For example, female 6-8week old C57B1/6 mice are obtained from Taconic (Germantown, NY). Groupsof mice are administered two subcutaneous (s.c.) injections at two siteson the back (upper and lower) of 0.1 ml myelin oligodentrocyteglycoprotein 35-55 (MOG35-55; 100 μg per injection; 200 μg per mouse(total 0.2 ml per mouse)), emulsified in Complete Freund's Adjuvant(CFA; 2-5mg killed mycobacterium tuberculosis H37Ra/ml emulsion).Approximately 1-2 hours after the above, mice are intraperitoneally(i.p.) injected with 200ng Pertussis toxin (PTx) in 0.1 ml PBS (2μg/ml). An additional IP injection of PTx is administered on day 2.Alternatively, an appropriate amount of an alternative myelin peptide(e.g. proteolipid protein (PLP)) is used to induce EAE. Some animalsserve as naive controls. EAE severity is assessed and a disability scoreis assigned daily beginning on day 4 according to methods known in theart (Mangalam et al. 2012).

Treatment with EVs is initiated at some point, either around the time ofimmunization or following EAE immunization. For example, EVs may beadministered at the same time as immunization (day 1), or they may beadministered upon the first signs of disability (e.g. limp tail), orduring severe EAE. EVs are administered at varied doses and at definedintervals. For example, some mice are intravenously injected with EVs at15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVsper mouse. While some mice receive EVs through i.v. injection, othermice may receive EVs through intraperitoneal (i.p.) injection,subcutaneous (s.c.) injection, nasal route administration, oral gavage,or other means of administration. Some mice may receive EVs every day(e.g. starting on day 1), while others may receive EVs at alternativeintervals (e.g. every other day, or once every three days). Additionalgroups of mice may receive some ratio of bacterial cells to EVs. Thebacterial cells may be live, dead, or weakened. The bacterial cells maybe harvested fresh (or frozen) and administered, or they may beirradiated or heat-killed prior to administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the EV administration. As with the EVs, bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, subcutaneous (s.c.)injection, or nasal route administration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) or EAE therapeutic(s) (e.g. anti-CD154, blockade of members ofthe TNF family, Vitamin D, or other treatment), and/or an appropriatecontrol (e.g. vehicle or control antibody) at various time points and ateffective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various timepoints, mice are sacrificed and sites of inflammation(e.g. brain and spinal cord), lymph nodes, or other tissues may beremoved for ex vivo histological, cytokine and/or flow cytometricanalysis using methods known in the art. For example, tissues aredissociated using dissociation enzymes according to the manufacturer'sinstructions. Cells are stained for analysis by flow cytometry usingtechniques known in the art. Staining antibodies can include anti-CD11c(dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII,anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzedinclude pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8,CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), andmacrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1,Gr-1, F4/80). In addition to immunophenotyping, serum cytokines areanalyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70,IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-lb, IFNy, GM-CSF, G-CSF,M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may becarried out on immune cells obtained from lymph nodes or other tissue,and/or on purified CD45+ central nervous system (CNS)-infiltrated immunecells obtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger (e.g. activated encephalitogenic T cells or re-injectionof EAE-inducing peptides). Mice are analyzed for susceptibility todisease and EAE severity following rechallenge.

Example 17 EVs in a Mouse Model of Collagen-Induced Arthritis (CIA)

Collagen-induced arthritis (CIA) is an animal model commonly used tostudy rheumatoid arthritis (RA), as described by Caplazi et al. (Mousemodels of rheumatoid arthritis. Veterinary Pathology. Sep. 1, 2015.52(5): 819-826) (see also Brand et al. Collagen-induced arthritis.Nature Protocols. 2007. 2: 1269-1275; Pietrosimone et al.Collagen-induced arthritis: a model for murine autoimmune arthritis. BioProtoc. 2015 Oct. 20; 5(20): e1626).

Among other versions of the CIA rodent model, one model involvesimmunizing HLA-DQ8 Tg mice with chick type II collagen as described byTaneja et al. (J. Immunology. 2007. 56: 69-78; see also Taneja et al. J.Immunology 2008. 181: 2869-2877; and Taneja et al. Arthritis Rheum.,2007. 56: 69-78). Purification of chick CII has been described by Tanejaet al. (Arthritis Rheum., 2007. 56: 69-78). Mice are monitored for CIAdisease onset and progression following immunization, and severity ofdisease is evaluated and “graded” as described by Wooley, J. Exp. Med.1981. 154: 688-700.

Mice are immunized for CIA induction and separated into varioustreatment groups. EVs are tested for their efficacy in CIA, either aloneor in combination with whole bacterial cells, with or without theaddition of other anti-inflammatory treatments.

Treatment with EVs is initiated either around the time of immunizationwith collagen or post-immunization. For example, in some groups, EVs maybe administered at the same time as immunization (day 1), or EVs may beadministered upon first signs of disease, or upon the onset of severesymptoms. EVs are administered at varied doses and at defined intervals.

For example, some mice are intravenously injected with EVs at 15, 20, or15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse.While some mice receive EVs through i.v. injection, other groups of micemay receive EVs through intraperitoneal (i.p.) injection, subcutaneous(s.c.) injection, nasal route administration, oral gavage, or othermeans of administration. Some mice may receive EVs every day (e.g.starting on day 1), while others may receive EVs at alternativeintervals (e.g. every other day, or once every three days). Additionalgroups of mice may receive some ratio of bacterial cells to EVs. Thebacterial cells may be live, dead, or weakened. The bacterial cells maybe harvested fresh (or frozen) and administered, or they may beirradiated or heat-killed prior to administration.

For example, some groups of mice may receive between 1×104 and 5×109bacterial cells in an administration separate from, or comingled with,the EV administration. As with the EVs, bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, subcutaneous (s.c.)injection, intradermal (i.d.) injection, or nasal route administration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) or CIA therapeutic(s) (e.g. anti-CD154, blockade of members ofthe TNF family, Vitamin D, or other treatment), and/or an appropriatecontrol (e.g. vehicle or control antibody) at various timepoints and ateffective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various timepoints, serum samples are obtained to assess levels ofanti-chick and anti-mouse CII IgG antibodies using a standard ELISA(Batsalova et al. Comparative analysis of collagen type II-specificimmune responses during development of collagen-induced arthritis in twoB10 mouse strains. Arthritis Res Ther. 2012. 14(6): R237). Also, somemice are sacrificed and sites of inflammation (e.g. synovium), lymphnodes, or other tissues may be removed for ex vivo histological,cytokine and/or flow cytometric analysis using methods known in the art.The synovium and synovial fluid are analyzed for plasma cellinfiltration and the presence of antibodies using techniques known inthe art. In addition, tissues are dissociated using dissociation enzymesaccording to the manufacturer's instructions to examine the profiles ofthe cellular infiltrates. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-lb, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+synovium-infiltrated immune cellsobtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger (e.g. activated re-injection with CIA-inducingpeptides). Mice are analyzed for susceptibility to disease and CIAseverity following rechallenge.

Example 18 EVs in a Mouse Model of Colitis

Dextran sulfate sodium (DSS)-induced colitis is a well-studied animalmodel of colitis, as reviewed by Randhawa et al. (A review onchemical-induced inflammatory bowel disease models in rodents. Korean JPhysiol Pharmacol. 2014. 18(4): 279-288; see also Chassaing et al.Dextran sulfate sodium (DSS)-induced colitis in mice. Curr ProtocImmunol. 2014 Feb. 4; 104: Unit 15.25).

EVs are tested for their efficacy in a mouse model of DSS-inducedcolitis, either alone or in combination with whole bacterial cells, withor without the addition of other anti-inflammatory agents.

Groups of mice are treated with DSS to induce colitis as known in theart (Randhawa et al. 2014; Chassaing et al. 2014; see also Kim et al.Investigating intestinal inflammation in DSS-induced model of IBD. J VisExp. 2012. 60: 3678). For example, male 6-8 week old C57B1/6 mice areobtained from Charles River Labs, Taconic, or other vendor. Colitis isinduced by adding 3% DSS (MP Biomedicals, Cat. #0260110) to the drinkingwater. Some mice do not receive DSS in the drinking water and serve asnaïve controls. Some mice receive water for five (5) days. Some mice mayreceive DSS for a shorter duration or longer than five (5) days. Miceare monitored and scored using a disability activity index known in theart based on weight loss (e.g. no weight loss (score 0); 1-5% weightloss (score 1); 5-10% weight loss (score 2)); stool consistency (e.g.normal (score 0); loose stool (score 2); diarrhea (score 4)); andbleeding (e.g. no blood (score 0), hemoccult positive (score 1);hemoccult positive and visual pellet bleeding (score 2); blood aroundanus, gross bleeding (score 4).

Treatment with EVs is initiated at some point, either on day 1 of DSSadministration, or sometime thereafter. For example, EVs may beadministered at the same time as DSS initiation (day 1), or they may beadministered upon the first signs of disease (e.g. weight loss ordiarrhea), or during the stages of severe colitis. Mice are observeddaily for weight, morbidity, survival, presence of diarrhea and/orbloody stool.

EVs are administered at varied doses and at defined intervals. Forexample, some mice are intravenously injected with EVs at 15, 20, or 15ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse.While some mice receive EVs through i.v. injection, other mice mayreceive EVs through intraperitoneal (i.p.) injection, subcutaneous(s.c.) injection, nasal route administration, oral gavage, or othermeans of administration. Some mice may receive EVs every day (e.g.starting on day 1), while others may receive EVs at alternativeintervals (e.g. every other day, or once every three days). Additionalgroups of mice may receive some ratio of bacterial cells to EVs. Thebacterial cells may be live, dead, or weakened. The bacterial cells maybe harvested fresh (or frozen) and administered, or they may beirradiated or heat-killed prior to administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the EV administration. As with the EVs, bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, or nasal routeadministration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) (e.g. anti-CD154, blockade of members of the TNF family, orother treatment), and/or an appropriate control (e.g. vehicle or controlantibody) at various timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some mice receive DSS without receiving antibioticsbeforehand.

At various timepoints, mice undergo video endoscopy using a small animalendoscope (Karl Storz Endoskipe, Germany) under isoflurane anesthesia.Still images and video are recorded to evaluate the extent of colitisand the response to treatment. Colitis is scored using criteria known inthe art. Fecal material is collected for study.

At various timepoints, mice are sacrificed and the colon, smallintestine, spleen, and lymph nodes (e.g. mesenteric lymph nodes) arecollected. Additionally, blood is collected into serum separation tubes.Tissue damage is assessed through histological studies that evaluate,but are not limited to, crypt architecture, degree of inflammatory cellinfiltration, and goblet cell depletion.

The gastrointestinal (GI) tract, lymph nodes, and/or other tissues maybe removed for ex vivo histological, cytokine and/or flow cytometricanalysis using methods known in the art. For example, tissues areharvested and may be dissociated using dissociation enzymes according tothe manufacturer's instructions. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+ GI tract-infiltrated immune cellsobtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger. Mice are analyzed for susceptibility to colitisseverity following rechallenge.

Example 19: EVs in a Mouse Model of Delayed-Type Hypersensitivity (DTH)

Delayed-type hypersensitivity (DTH) is an animal model of atopicdermatitis (or allergic contact dermatitis), as reviewed by Petersen etal. (In vivo pharmacological disease models for psoriasis and atopicdermatitis in drug discovery. Basic & Clinical Pharm & Toxicology. 2006.99(2): 104-115; see also Irving C. Allen (ed.) Mouse Models of InnateImmunity: Methods and Protocols, Methods in Molecular Biology, 2013.vol. 1031, DOI 10.1007/978-1-62703-481-4_13). It can be induced in avariety of mouse and rat strains using various haptens or antigens, forexample an antigen emulsified with an adjuvant. DTH is characterized bysensitization as well as an antigen-specific T cell-mediated reactionthat results in erythema, edema, and cellular infiltration—especiallyinfiltration of antigen presenting cells (APCs), eosinophils, activatedCD4+T cells, and cytokine-expressing Th2 cells.

Generally, mice are primed with an antigen administered in the contextof an adjuvant (e.g. Complete Freund's Adjuvant) in order to induce asecondary (or memory) immune response measured by swelling andantigen-specific antibody titer.

EVs are tested for their efficacy in the mouse model of DTH, eitheralone or in combination with whole bacterial cells, with or without theaddition of other anti-inflammatory treatments. For example, 6-8 weekold C57B1/₆ mice are obtained from Taconic (Germantown, NY), or othervendor. Groups of mice are administered four subcutaneous (s.c.)injections at four sites on the back (upper and lower) of antigen (e.g.Ovalbumin (OVA)) in an effective dose (50 μl total volume per site). Fora DTH response, animals are injected intradermally (i.d.) in the earsunder ketamine/xylazine anesthesia (approximately 50 mg/kg and 5 mg/kg,respectively). Some mice serve as control animals. Some groups of miceare challenged with 10 μl per ear (vehicle control (0.01% DMSO insaline) in the left ear and antigen (21.2 ug (12nmol) in the right ear)on day 8. To measure ear inflammation, the ear thickness of manuallyrestrained animals is measured using a Mitutoyo micrometer. The earthickness is measured before intradermal challenge as the baseline levelfor each individual animal. Subsequently, the ear thickness is measuredtwo times after intradermal challenge, at approximately 24 hours and 48hours (i.e. days 9 and 10).

Treatment with EVs is initiated at some point, either around the time ofpriming or around the time of DTH challenge. For example, EVs may beadministered at the same time as the subcutaneous injections (day 0), orthey may be administered prior to, or upon, intradermal injection. EVsare administered at varied doses and at defined intervals. For example,some mice are intravenously injected with EVs at 15, 20, or 15 μg/mouse.Other mice may receive 25, 50, or 100 mg of EVs per mouse. While somemice receive EVs through i.v. injection, other mice may receive EVsthrough intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection,nasal route administration, oral gavage, topical administration,intradermal (i.d.) injection, or other means of administration. Somemice may receive EVs every day (e.g. starting on day 0), while othersmay receive EVs at alternative intervals (e.g. every other day, or onceevery three days). Additional groups of mice may receive some ratio ofbacterial cells to EVs. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the EV administration. As with the EVs, bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, i.d. injection,topical administration, or nasal route administration.

Mice were injected with KLH and CFA i.d at 4 locations along the back(50ug per mouse of KLH prepared in a 1:1 ratio with CFA in a totalvolume of 50ul per site). Mice were dosed for 9 days as follows; 1) oraladministration of anaerobic PBS (vehicle); 2) oral administration of10mg Prevotella histicola; 3) oral administration of 100ug P.histicola-derived EVs; 4) i.p. administration of PBS; 5) i.p.administration of Dexamethasone (positive control); and 6) i.p.administration of 10ug Prevotella histicola-derived EVs. For the EVs,total protein was measured using Bio-rad assays (Cat# 5000205) performedper manufacturer's instructions. At 24 and 48 hours post-challenge with10ug of KLH (10ul volume), groups receiving Prevotella histicola (livecells) or Prevotella histicola-derived EVs, in both the oral and i.padministration groups, exhibited less inflammation than the vehiclegroups (FIGS. 3A and 3B). A dose dependent DTH response following i.p.injection of Prevotella histicola-derived EVs at 10 μg, 3 μg, 1 μg, and0.1 μg was observed in reducing antigen-specific ear swelling (earthickness) 48 hours after antigen challenge in a KLH-based delayed typehypersensitivity mouse model (FIG. 3C).

Mice were injected with KLH and CFA i.d at 4 locations along the back(50 μg per mouse of KLH prepared in a 1:1 ratio with CFA in a totalvolume of 50 μl per site). Mice were dosed for 9 days as follows; 1)oral administration of anaerobic PBS (vehicle); 2) oral administrationof 10 mg Prevotella histicola; 3) oral administration of 1×10⁹ CFUPrevotella histicola biomass; 4) oral administration of 2.09×10⁸ CFUPrevotella melanogenica biomass ; 5) oral administration of 100ug P.histicola-derived EVs; 6) oral administration of 100ug P.melanogenica-derived EVs; and 7) i.p. administration of Dexamethasone(positive control). For the EVs, total protein was measured usingBio-rad assays (Cat# 5000205) performed per manufacturer's instructions.At 24 and 48 hours post-challenge with bug of KLH (10ul volume), groupsreceiving Prevotella histicola (live cells) or Prevotellahisticola-derived EVs exhibited less inflammation than the vehiclegroups (FIGS. 8A and 8B). At 24 and 48 hours post-challenge with bug ofKLH (10ul volume), the group receiving Prevotella melanogenica-derivedEVs exhibited less inflammation than the vehicle groups and the groupreceiving Prevotella melanogenica (live cells) (FIGS. 8A and 8B).

The test formulations were prepared for KLH-based delayed typehypersensitivity model. The DTH model provides an in vivo mechanism tostudy the cell-mediated immune response, and resulting inflammation,following exposure to a specific antigen to which the mice have beensensitized. Several variations of the DTH model have been used and arewell known in the art (Irving C. Allen (ed.). Mouse Models of InnateImmunity: Methods and Protocols, Methods in Molecular Biology. Vol.1031, DOI 10.1007/978-1-62703-481-4_13, Springer Science+Business Media,LLC 2013). For example, the emulsion of Keyhole Limpet Hemocyanin (KLH)and Complete Freund's Adjuvant (CFA) are prepared freshly on the day ofimmunization (day 0). To this end, 8 mg of KLH powder is weighed and isthoroughly re-suspended in 16 mL saline. An emulsion is prepared bymixing the KLH/saline with an equal volume of CFA solution (e.g. 10 mLKLH/saline+10 mL CFA solution) using syringes and a luer lock connector.KLH and CFA is mixed vigorously for several minutes to form awhite-colored emulsion to obtain maximum stability. A drop test isperformed to check if a homogenous emulsion is obtained, mixing iscontinued until an intact drop remains visible in the water.

On day 0, C57B1/6J female mice, approximately 7 weeks old, were primedwith KLH antigen in CFA by subcutaneous immunization (4 sites, 50 μL persite).

Dexamethasone, a corticosteroid, is a known anti-inflammatory thatameliorates DTH reactions in mice, and serves as a positive control forsuppressing inflammation in this model (Taube and Carlsten, Action ofdexamethasone in the suppression of delayed-type hypersensitivity inreconstituted SCID mice. Inflamm Res. 2000. 49(10): 548-52). For thepositive control group, a stock solution of 17 mg/mL of Dexamethasonewas prepared on Day 0 by diluting 6.8 mg Dexamethasone in 400 μL96%ethanol. For each day of dosing, a working solution is prepared bydiluting the stock solution 100× in sterile PBS to obtain a finalconcentration of 0.17 mg/mL in a septum vial for intraperitoneal dosing.Dexamethasone-treated mice received 100 μL Dexamethasone i.p. (5 mL/kgof a 0.17 mg/mL solution). Frozen sucrose served as the negative control(vehicle). Veillonella Strains were dosed at 1×10¹⁰ CFU p.o. daily.Dexamethasone (positive control), vehicle (negative control), andBifidobacterium animalis lactis (10 mg powder) were dosed daily.

On day 8, mice were challenged intradermally (i.d.) with 10 μg KLH insaline (in a volume of 10 μL) in the left ear. Inflammatory responseswere measured using methods known in the art. Ear pinna thickness wasmeasured at 24 hours following antigen challenge (FIG. 11). Asdetermined by ear thickness, Veillonella Strains were efficacious atsuppressing inflammation compared to mice that received vehicle alone(comparable to Dexamethasone treatment).

The efficacy of Veillonella strains may be studied further using variedtiming and varied doses. For instance, treatment with a Veillonellabacterial composition may be initiated at some point, either around thetime of priming or around the time of DTH challenge. For example,Veillonella (1×10⁹ CFU per mouse per day) may be administered at thesame time as the subcutaneous injections (day 0), or administered priorto, or upon, intradermal injection. Veillonella strains may beadministered at varied doses and at defined intervals, and in variouscombinations. For example, some mice are intravenously injected withVeillonella at a range of between 1×10⁴ and 5×10⁹ bacterial cells permouse. Some mice receive a mixture of Strains. While some mice willreceive a Veillonella through i.v. injection, other mice may receive aVeillonella through intraperitoneal (i.p.) injection, subcutaneous(s.c.) injection, nasal route administration, oral gavage, topicaladministration, intradermal (i.d.) injection, or other means ofadministration. Some mice may receive a Veillonella every day (e.g.starting on day 0), while others may receive a Veillonella atalternative intervals (e.g. every other day, or once every three days).The bacterial cells may be live, dead, or weakened. The bacterial cellsmay be harvested fresh (or frozen) and administered, or they may beirradiated or heat-killed prior to administration.

Some groups of mice may be treated with anti-inflammatory agent(s) (e.g.anti-CD154, blockade of members of the TNF family, or other treatment),and/or an appropriate control (e.g. vehicle or control antibody) atvarious timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5g/L), ampicillin (1.0g/L), gentamicin(1.0g/L) and amphotericin B (0.2g/L) are added to the drinking water,and antibiotic treatment is halted at the time of treatment or a fewdays prior to treatment. Some immunized mice are treated withoutreceiving antibiotics.

At various timepoints, serum samples are taken. Other groups of mice aresacrificed and lymph nodes, spleen, mesenteric lymph nodes (MLN), thesmall intestine, colon, and other tissues may be removed for histologystudies, ex vivo histological, cytokine and/or flow cytometric analysisusing methods known in the art. Some mice are exsanguinated from theorbital plexus under 02/CO2 anesthesia and ELISA assays performed.

Tissues may be dissociated using dissociation enzymes according to themanufacturer's instructions. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-lb, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+ infiltrated immune cells obtainedex vivo. Finally, immunohistochemistry is carried out on various tissuesections to measure T cells, macrophages, dendritic cells, andcheckpoint molecule protein expression.

Mice were primed and challenged with KLH as described above and,following measurement of the ear swelling at 48 hours, mice weresacrificed.

Ears were removed from the sacrificed animals and placed in coldEDTA-free protease inhibitor cocktail (Roche). Ears were homogenizedusing bead disruption and supernatants analyzed for IL-1β by Luminex kit(EMD Millipore) as per manufacturer's instructions. Mice that weretreated with 10 μg P. histicola EVs (i.p.) showed levels of IL-1βcomparable to that seen in the Dexamethasone group (positive control).(FIG. 3D). P. histicola-derived EVs are capable of suppressingpro-inflammatory cytokines.

In addition, cervical lymph nodes were dissociated through a cellstrainer, washed, and stained for FoxP3 (PE-FJK-16s) and CD25(FITC-PC61.5) using methods known in the art (FIG. 3E). Mice that weretreated with 10 μg P. histicola EVs (i.p.) showed an increase in Tregsin the cervical lymph nodes relative to naïve mice (negative control),and comparable to the Dexamethasone group (positive control). P.histicola-derived EVs are capable of inducing Tregs in draining lymphnodes of challenged mice.

In order to examine the impact and longevity of DTH protection, ratherthan being sacrificed, some mice may be rechallenged with thechallenging antigen (e.g. OVA). Mice are analyzed for susceptibility toDTH and severity of response.

Example 20 EVs in a Mouse Model of Type 1 Diabetes (T1D)

Type 1 diabetes (T1D) is an autoimmune disease in which the immunesystem targets the islets of Langerhans of the pancreas, therebydestroying the body's ability to produce insulin.

There are various models of animal models of T1D, as reviewed by Belleet al. (Mouse models for type 1 diabetes. Drug Discov Today Dis Models.2009; 6(2): 41-45; see also Aileen J F King. The use of animal models indiabetes research. Br J Pharmacol. 2012 June; 166(3): 877-894. There aremodels for chemically-induced T1D, pathogen-induced T1D, as well asmodels in which the mice spontaneously develop T1D.

EVs are tested for their efficacy in a mouse model of T1D, either aloneor in combination with whole bacterial cells, with or without theaddition of other anti-inflammatory treatments.

Depending on the method of T1D induction and/or whether T1D developmentis spontaneous, treatment with EVs is initiated at some point, eitheraround the time of induction or following induction, or prior to theonset (or upon the onset) of spontaneously-occurring T1D. EVs areadministered at varied doses and at defined intervals. For example, somemice are intravenously injected with EVs at 15, 20, or 15 ug/mouse.Other mice may receive 25, 50, or 100 mg of EVs per mouse. While somemice receive EVs through i.v. injection, other mice may receive EVsthrough intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection,nasal route administration, oral gavage, or other means ofadministration. Some mice may receive EVs every day, while others mayreceive EVs at alternative intervals (e.g. every other day, or onceevery three days). Additional groups of mice may receive some ratio ofbacterial cells to EVs. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the EV administration. As with the EVs, bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, or nasal routeadministration.

Some groups of mice may be treated with additional treatments and/or anappropriate control (e.g. vehicle or control antibody) at varioustimepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

Blood glucose is monitored biweekly prior to the start of theexperiment. At various timepoints thereafter, nonfasting blood glucoseis measured. At various timepoints, mice are sacrificed and site thepancreas, lymph nodes, or other tissues may be removed for ex vivohistological, cytokine and/or flow cytometric analysis using methodsknown in the art. For example, tissues are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-lb, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified tissue-infiltratingimmune cells obtained ex vivo. Finally, immunohistochemistry is carriedout on various tissue sections to measure T cells, macrophages,dendritic cells, and checkpoint molecule protein expression. Antibodyproduction may also be assessed by ELISA.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger, or assessed for susceptibility to relapse. Mice areanalyzed for susceptibility to diabetes onset and severity followingrechallenge (or spontaneously-occurring relapse).

Example 21 EVs in a Mouse Model of Primary Sclerosing Cholangitis (PSC)

Primary Sclerosing Cholangitis (PSC) is a chronic liver disease thatslowly damages the bile ducts and leads to end-stage cirrhosis. It isassociated with inflammatory bowel disease (IBD).

There are various animal models for PSC, as reviewed by Fickert et al.(Characterization of animal models for primary sclerosing cholangitis(PSC). J Hepatol. 2014 Jun. 60(6): 1290-1303; see also Pollheimer andFickert. Animal models in primary biliary cirrhosis and primarysclerosing cholangitis. Clin Rev Allergy Immunol. 2015 Jun. 48(2-3):207-17). Induction of disease in PSC models includes chemical induction(e.g. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-inducedcholangitis), pathogen-induced (e.g. Cryptosporidium parvum),experimental biliary obstruction (e.g. common bile duct ligation(CBDL)), and transgenic mouse model of antigen-driven biliary injury(e.g. Ova-Bil transgenic mice). For example, bile duct ligation isperformed as described by Georgiev et al. (Characterization oftime-related changes after experimental bile duct ligation. Br J Surg.2008. 95(5): 646-56), or disease is induced by DCC exposure as describedby Fickert et al. (A new xenobiotic-induced mouse model of sclerosingcholangitis and biliary fibrosis. Am J Path. Vol 171(2): 525-536.

EVs are tested for their efficacy in a mouse model of PSC, either aloneor in combination with whole bacterial cells, with or without theaddition of some other therapeutic agent.

DCC-induced Cholangitis

For example, 6-8 week old C57b¹/₆ mice are obtained from Taconic orother vendor. Mice are fed a 0.1% DCC-supplemented diet for variousdurations. Some groups receive DCC-supplement food for 1 week, othersfor 4 weeks, others for 8 weeks. Some groups of mice may receive aDCC-supplemented diet for a length of time and then be allowed torecover, thereafter receiving a normal diet. These mice may be studiedfor their ability to recover from disease and/or their susceptibility torelapse upon subsequent exposure to DCC. Treatment with EVs is initiatedat some point, either around the time of DCC-feeding or subsequent toinitial exposure to DCC. For example, EVs may be administered on day 1,or they may be administered sometime thereafter. EVs are administered atvaried doses and at defined intervals. For example, some mice areintravenously injected with EVs at 15, 20, 0r 15 ug/mouse. Other micemay receive 25, 50, 100 mg of EVs per mouse. While some mice receive EVsthrough i.v. injection, other mice may receive EVs through i.p.injection, subcutaneous (s.c.) injection, nasal route administration,oral gavage, or other means of administration. Some mice may receive EVsevery day (e.g. starting on day 1), while others may receive EVs atalternative intervals (e.g. every other day, or once every three days).Additional groups of mice may receive some ratio of bacterial cells toEVs. The bacterial cells may be live, dead, or weakened. The bacterialcells may be harvested fresh (or frozen), and administered, or they maybe irradiated or heat-killed prior to administration. For example, somegroups of mice may receive between 1×10⁴ and 5×10⁹ bacterial cells in anadministration separate from, or comingled with, the EV administration.As with EVs, bacterial cell administration may be varied by route ofadministration, dose, and schedule. This can include oral gavage, i.v.injection, i.p. injection, or nasal route administration. Some groups ofmice may be treated with additional agents and/or an appropriate control(e.g. vehicle or antibody) at various timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5g/L), ampicillin (1.0g/L), gentamicin(1.0g/L) and amphotericin B (0.2g/L) are added to the drinking water,and antibiotic treatment is halted at the time of treatment or a fewdays prior to treatment. Some immunized mice are treated withoutreceiving antibiotics. At various timepoints, serum samples are analyzedfor ALT, AP, bilirubin, and serum bile acid (BA) levels.

At various timepoints, mice are sacrificed, body and liver weight arerecorded, and sites of inflammation (e.g. liver, small and largeintestine, spleen), lymph nodes, or other tissues may be removed for exvivo histolomorphological characterization, cytokine and/or flowcytometric analysis using methods known in the art (see Fickert et al.Characterization of animal models for primary sclerosing cholangitis(PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts arestained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues arestained for histological examination, while others are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11 c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80), as well asadhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-lb, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+ bileduct-infiltrated immune cells obtained ex vivo.

Liver tissue is prepared for histological analysis, for example, usingSirius-red staining followed by quantification of the fibrotic area. Atthe end of the treatment, blood is collected for plasma analysis ofliver enzymes, for example, AST or ALT, and to determine Bilirubinlevels. The hepatic content of Hydroxyproline can be measured usingestablished protocols. Hepatic gene expression analysis of inflammationand fibrosis markers may be performed by qRT-PCR using validatedprimers. These markers may include, but are not limited to, MCP-1,alpha-SMA, Coll1al, and TIMP-. Metabolite measurements may be performedin plasma, tissue and fecal samples using established metabolomicsmethods. Finally, immunohistochemistry is carried out on liver sectionsto measure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with DCC ata later time. Mice are analyzed for susceptibility to cholangitis andcholangitis severity following rechallenge.

BDL—induced Cholangitis

Alternatively, EVs are tested for their efficacy in BDL-inducedcholangitis. For example, 6-8 week old C57B1/6J mice are obtained fromTaconic or other vendor. After an acclimation period the mice aresubjected to a surgical procedure to perform a bile duct ligation (BDL).Some control animals receive a sham surgery. The BDL procedure leads toliver injury, inflammation and fibrosis within 7-21 days.

Treatment with EVs is initiated at some point, either around the time ofsurgery or some time following the surgery. EVs are administered atvaried doses and at defined intervals. For example, some mice areintravenously injected with EVs at 15, 20, or 15 ug/mouse. Other micemay receive 25, 50, or 100 mg of EVs per mouse. While some mice receiveEVs through i.v. injection, other mice may receive EVs through i.p.injection, subcutaneous (s.c.) injection, nasal route administration,oral gavage, or other means of administration. Some mice receive EVsevery day (e.g. starting on day 1), while others may receive EVs atalternative intervals (e.g. every other day, or once every three days).Additional groups of mice may receive some ratio of bacterial cells toEVs. The bacterial cells may be live, dead, or weakened. They bacterialcells may be harvested fresh (or frozen), and administered, or they maybe irradiated or heat-killed prior to administration. For example, somegroups of mice may receive between 1×10⁴ and 5×10⁹ bacterial cells in anadministration separate from, or comingled with, the EV administration.As with EVs, bacterial cell administration may be varied by route ofadministration, dose, and schedule. This can include oral gavage, i.v.injection, i.p. injection, or nasal route administration. Some groups ofmice may be treated with additional agents and/or an appropriate control(e.g. vehicle or antibody) at various timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5g/L), ampicillin (1.0g/L), gentamicin(1.0g/L) and amphotericin B (0.2g/L) are added to the drinking water,and antibiotic treatment is halted at the time of treatment or a fewdays prior to treatment. Some immunized mice are treated withoutreceiving antibiotics. At various timepoints, serum samples are analyzedfor ALT, AP, bilirubin, and serum bile acid (BA) levels.

At various timepoints, mice are sacrificed, body and liver weight arerecorded, and sites of inflammation (e.g. liver, small and largeintestine, spleen), lymph nodes, or other tissues may be removed for exvivo histolomorphological characterization, cytokine and/or flowcytometric analysis using methods known in the art (see Fickert et al.Characterization of animal models for primary sclerosing cholangitis(PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts arestained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues arestained for histological examination, while others are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80), as well asadhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-lb, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+bileduct-infiltrated immune cells obtained ex vivo.

Liver tissue is prepared for histological analysis, for example, usingSirius-red staining followed by quantification of the fibrotic area. Atthe end of the treatment, blood is collected for plasma analysis ofliver enzymes, for example, AST or ALT, and to determine Bilirubinlevels. The hepatic content of Hydroxyproline can be measured usingestablished protocols. Hepatic gene expression analysis of inflammationand fibrosis markers may be performed by qRT-PCR using validatedprimers. These markers may include, but are not limited to, MCP-1,alpha-SMA, Coll1a , and TIMP-. Metabolite measurements may be performedin plasma, tissue and fecal samples using established metabolomicsmethods. Finally, immunohistochemistry is carried out on liver sectionsto measure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be analyzed for recovery.

Example 22 EVs in a Mouse Model of Nonalcoholic Steatohepatitis (NASH)

Nonalcoholic Steatohepatitis (NASH) is a severe form of NonalcoholicFatty Liver Disease (NAFLD), where buildup of hepatic fat (steatosis)and inflammation lead to liver injury and hepatocyte cell death(ballooning).

There are various animal models of NASH, as reviewed by Ibrahim et al.(Animal models of nonalcoholic steatohepatitis: Eat, Delete, andInflame. Dig Dis Sci. 2016 May. 61(5): 1325-1336; see also Lau et al.Animal models of non-alcoholic fatty liver disease: current perspectivesand recent advances 2017 January. 241(1): 36-44).

EVs are tested for their efficacy in a mouse model of NASH, either aloneor in combination with whole bacterial cells, with or without theaddition of another therapeutic agent. For example, 8-10 week oldC57B1/6J mice, obtained from Taconic (Germantown, N.Y.), or othervendor, are placed on a methionine choline deficient (MCD) diet for aperiod of 4-8 weeks during which NASH features develop, includingsteatosis, inflammation, ballooning and fibrosis.

Prevotella histicola bacterial cells and P. histicola-derived EVs aretested for their efficacy in a mouse model of NASH, either alone or incombination with each other, in varying proportions, with or without theaddition of another therapeutic agent. For example, 8 week old C57B1/6Jmice, obtained from Charles River (France), or other vendor, wereacclimated for a period of 5 days, randomized intro groups of 10 micebased on body weight, and placed on a methionine choline deficient (MCD)diet for example A02082002B from Research Diets (USA), for a period of 4weeks during which NASH features developed, including steatosis,inflammation, ballooning and fibrosis. Control chow mice were fed anormal chow diet, for example RM1 (E) 801492 from SDS Diets (UK).Control chow, MCD diet, and water were provided ad libitum.

Treatment with frozen, live P. histicola was initiated in day 1 of MCDdiet for some mice and continued for 28 consecutive days. Some MCD dietmice were administered bacterial cells through daily oral gavage of 100μl of a suspension containing 1.47×10⁹ bacterial cells. Control chow andsome MCD diet mice remained untreated, while some MCD diet mice wereadministered daily with a vehicle solution, through daily oral gavage,for 28 days. Some MCD diet mice were administered the reference compoundand FXR agonist, obeticholic acid (OCA; positive control), at a dose of30 mg/kg, through daily oral gavage, for 28 days. At the end of thetreatment (day 28), mice are sacrificed and liver, small intestine,lumenal contents, blood, and feces, were removed for ex vivohistological, biochemical, molecular or cytokine and/or flow cytometryanalysis using methods known in the art. For example, 0.5 cm³ liversamples were stored in formalin for 24 hours and then in ethanol at 4°C., prior to hematoxylin/eosin (H&E) and Sirius Red staining, anddetermination of NASH activity score (NAS). Histological analysis andscoring was conducted at Histalim (Montpelier, France) in a blindedmanner. Slides containing one hepatic lobe section stained with eitherH&E or Sirius red were digitized using a NanoZoomer and visualized usingNDP viewer, both from Hamamatsu (Japan). Each section was evaluated andscored individually. A NAS scoring system adapted from Kleiner et al.(Design and validation of a histological scoring system for nonalcoholicfatty liver disease. Hepatology. 2005 June 41(6): 1313-1321) was used todetermine the degree of steatosis (scored 0-3), lobular inflammation(scored 0-3), hepatocyte ballooning (scored 0-3), and fibrosis (scored0-4). An individual mouse NAS score was calculated by summing the scorefor steatosis, inflammation, ballooning, and fibrosis (scored 0-13). Inaddition, the levels of plasma AST and ALT were determined using aPentra 400 instrument from Horiba (USA), according to manufacturer'sinstructions. The levels of hepatic total cholesterol, triglycerides,fatty acids, alanine aminotransferase, and aspartate aminotransferasewere also determined using methods known in the art.

In mice receiving the MCD (NASH-inducing) diet, orally administered P.histicola was efficacious in reducing the NAS score compared to vehicleand no treatment groups (negative controls) (FIG. 4). P. histicolareduced steatosis (FIG. 5A), inflammation (FIG. 5B and FIG. 5C), andballooning (FIG. 5D), as well as hepatic total cholesterol (FIG. 6). P.histicola also reduced the fibrosis score in treated mice (FIG. 7A andFIG. 7B).

FIG. 9A shows P. histicola effect on hepatic free fatty acids in micethat were fed an MCD diet, FIG. 9B shows P. histicola effect on hepatictotal cholesterol in mice that were fed an MCD diet, FIG. 9C shows P.histicola effect on hepatic triglycerides in mice that were fed an MCDdiet, FIG. 9D shows P. histicola and P. melanogenica effect on alanineaminotransferase in mice that were fed an MCD diet, FIG. 9E shows P.histicola and P. melanogenica effect on aspartate aminotransferase inmice that were fed an MCD diet.

In mice receiving the MCD (NASH-inducing) diet, orally administered P.histicola and P. melanogenica was efficacious in reducing the NAS scorecompared to vehicle and no treatment groups (negative controls) (FIGS.10A and 10B).

In other studies, hepatic gene expression analysis of inflammation,fibrosis, steatosis, ER stress, or oxidative stress markers may beperformed by qRT-PCR using validated primers. These markers may include,but are not limited to, IL-1β, TNF-α, MCP-1, α-SMA, Coll1al, CHOP, andNRF2.

Treatment with EVs is initiated at some point, either at the beginningof the diet, or at some point following diet initiation (for example,one week after). For example, EVs may be administered starting in thesame day as the initiation of the MCD diet. EVs are administered atvaried doses and at defined intervals. For example, some mice areintravenously injected with EVs at 15, 20, or 15 ug/mouse. Other micemay receive 25, 50, or 100 mg of EVs per mouse. While some mice receiveEVs through i.v. injection, other mice may receive EVs throughintraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasalroute administration, oral gavage, or other means of administration.Some mice may receive EVs every day (e.g. starting on day 1), whileothers may receive EVs at alternative intervals (e.g. every other day,or once every three days). Additional groups of mice may receive someratio of bacterial cells to EVs. The bacterial cells may be live, dead,or weakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the EV administration. As with the EVs, bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, or nasal routeadministration. Some groups of mice may be treated with additional NASHtherapeutic(s) (e.g., FXR agonists, PPAR agonists, CCR2/5 antagonists orother treatment) and/or appropriate control at various timepoints andeffective doses.

At various timepoints and/or at the end of the treatment, mice aresacrificed and liver, intestine, blood, feces, or other tissues may beremoved for ex vivo histological, biochemical, molecular or cytokineand/or flow cytometry analysis using methods known in the art. Forexample, liver tissues are weighed and prepared for histologicalanalysis, which may comprise staining with H&E, Sirius Red, anddetermination of NASH activity score (NAS). At various timepoints, bloodis collected for plasma analysis of liver enzymes, for example, AST orALT, using standards assays. In addition, the hepatic content ofcholesterol, triglycerides, or fatty acid acids can be measured usingestablished protocols. Hepatic gene expression analysis of inflammation,fibrosis, steatosis, ER stress, or oxidative stress markers may beperformed by qRT-PCR using validated primers. These markers may include,but are not limited to, IL-6, MCP-1, alpha-SMA, Coll1al, CHOP, and NRF2.Metabolite measurements may be performed in plasma, tissue and fecalsamples using established biochemical and mass-spectrometry-basedmetabolomics methods. Serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+bileduct-infiltrated immune cells obtained ex vivo. Finally,immunohistochemistry is carried out on liver or intestine sections tomeasure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be analyzed for recovery.

Example 23 EVs in a Mouse Model of Psoriasis

Psoriasis is a T-cell-mediated chronic inflammatory skin disease.So-called “plaque-type” psoriasis is the most common form of psoriasisand is typified by dry scales, red plaques, and thickening of the skindue to infiltration of immune cells into the dermis and epidermis.Several animal models have contributed to the understanding of thisdisease, as reviewed by Gudjonsson et al. (Mouse models of psoriasis. JInvest Derm. 2007. 127: 1292-1308; see also van der Fits et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediatedvia the IL-23/IL-17 axis. J. Immunol. 2009 May 1. 182(9): 5836-45).

Psoriasis can be induced in a variety of mouse models, including thosethat use transgenic, knockout, or xenograft models, as well as topicalapplication of imiquimod (IMQ), a TLR7/8 ligand.

EVs are tested for their efficacy in the mouse model of psoriasis,either alone or in combination with whole bacterial cells, with orwithout the addition of other anti-inflammatory treatments. For example,6-8 week old C57B1/6 or Balb/c mice are obtained from Taconic(Germantown, N.Y.), or other vendor. Mice are shaved on the back and theright ear. Groups of mice receive a daily topical dose of 62.5 mg ofcommercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals). Thedose is applied to the shaved areas for 5 or 6 consecutive days. Atregular intervals, mice are scored for erythema, scaling, and thickeningon a scale from 0 to 4, as described by van der Fits et al. (2009). Miceare monitored for ear thickness using a Mitutoyo micrometer.

Treatment with EVs is initiated at some point, either around the time ofthe first application of IMQ, or something thereafter. For example, EVsmay be administered at the same time as the subcutaneous injections (day0), or they may be administered prior to, or upon, application. EVs areadministered at varied doses and at defined intervals. For example, somemice are intravenously injected with EVs at 15, 20, or 15 ug/mouse.Other mice may receive 25, 50, or 100 mg of EVs per mouse. While somemice receive EVs through i.v. injection, other mice may receive EVsthrough intraperitoneal (i.p.) injection, nasal route administration,oral gavage, topical administration, intradermal (i.d.) injection,subcutaneous (s.c.) injection, or other means of administration. Somemice may receive EVs every day (e.g. starting on day 0), while othersmay receive EVs at alternative intervals (e.g. every other day, or onceevery three days). Additional groups of mice may receive some ratio ofbacterial cells to EVs. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the EV administration. As with the EVs, bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, i.d. injection,s.c. injection, topical administration, or nasal route administration.

Some groups of mice may be treated with anti-inflammatory agent(s) (e.g.anti-CD154, blockade of members of the TNF family, or other treatment),and/or an appropriate control (e.g. vehicle or control antibody) atvarious timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5g/L), ampicillin (1.0g/L), gentamicin(1.0g/L) and amphotericin B (0.2g/L) are added to the drinking water,and antibiotic treatment is halted at the time of treatment or a fewdays prior to treatment. Some immunized mice are treated withoutreceiving antibiotics.

At various timepoints, samples from back and ear skin are taken forcryosection staining analysis using methods known in the art. Othergroups of mice are sacrificed and lymph nodes, spleen, mesenteric lymphnodes (MLN), the small intestine, colon, and other tissues may beremoved for histology studies, ex vivo histological, cytokine and/orflow cytometric analysis using methods known in the art. Some tissuesmay be dissociated using dissociation enzymes according to themanufacturer's instructions. Cryosection samples, tissue samples, orcells obtained ex vivo are stained for analysis by flow cytometry usingtechniques known in the art. Staining antibodies can include anti-CD11c(dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII,anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzedinclude pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8,CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), andmacrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1,Gr-1, F4/80). In addition to immunophenotyping, serum cytokines areanalyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70,IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF,M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may becarried out on immune cells obtained from lymph nodes or other tissue,and/or on purified CD45+skin-infiltrated immune cells obtained ex vivo.Finally, immunohistochemistry is carried out on various tissue sectionsto measure T cells, macrophages, dendritic cells, and checkpointmolecule protein expression.

In order to examine the impact and longevity of psoriasis protection,rather than being sacrificed, some mice may be studied to assessrecovery, or they may be rechallenged with IMQ. The groups ofrechallenged mice are analyzed for susceptibility to psoriasis andseverity of response.

Example 24 Manufacturing Conditions

Enriched media is used to grow and prepare the bacterium for in vitroand in vivo use. For example, media may contain sugar, yeast extracts,plant based peptones, buffers, salts, trace elements, surfactants,anti-foaming agents, and vitamins. Composition of complex componentssuch as yeast extracts and peptones may be undefined or partiallydefined (including approximate concentrations of amino acids, sugarsetc.). Microbial metabolism may be dependent on the availability ofresources such as carbon and nitrogen. Various sugars or other carbonsources may be tested. Alternatively, media may be prepared and theselected bacterium grown as shown by Saarela et al., J. AppliedMicrobiology. 2005. 99: 1330-1339, which is hereby incorporated byreference. Influence of fermentation time, cryoprotectant andneutralization of cell concentrate on freeze-drying survival, storagestability, and acid and bile exposure of the selected bacterium producedwithout milk-based ingredients.

At large scale, the media is sterilized. Sterilization may be by UltraHigh Temperature (UHT) processing. The UHT processing is performed atvery high temperature for short periods of time. The UHT range may befrom 135-180° C. For example, the medium may be sterilized from between10 to 30 seconds at 135° C.

Inoculum can be prepared in flasks or in smaller bioreactors and growthis monitored. For example, the inoculum size may be betweenapproximately 0.5 and 3% of the total bioreactor volume. Depending onthe application and need for material, bioreactor volume can be at least2L, 10L, 80L, 100L, 250L, 1000L, 2500L, 5000L, 10,000L.

Before the inoculation, the bioreactor is prepared with medium atdesired pH, temperature, and oxygen concentration. The initial pH of theculture medium may be different that the process set-point. pH stressmay be detrimental at low cell centration; the initial pH could bebetween pH 7.5 and the process set-point. For example, pH may be setbetween 4.5 and 8.0. During the fermentation, the pH can be controlledthrough the use of sodium hydroxide, potassium hydroxide, or ammoniumhydroxide. The temperature may be controlled from 25° C. to 45° C., forexample at 37° C. Anaerobic conditions are created by reducing the levelof oxygen in the culture broth from around 8 mg/L to 0 mg/L. Forexample, nitrogen or gas mixtures (N2, CO2, and H2) may be used in orderto establish anaerobic conditions. Alternatively, no gases are used andanaerobic conditions are established by cells consuming remaining oxygenfrom the medium. Depending on strain and inoculum size, the bioreactorfermentation time can vary. For example, fermentation time can vary fromapproximately 5 hours to 48 hours.

Reviving microbes from a frozen state may require specialconsiderations. Production medium may stress cells after a thaw; aspecific thaw medium may be required to consistently start a seed trainfrom thawed material. The kinetics of transfer or passage of seedmaterial to fresh medium, for the purposes of increasing the seed volumeor maintaining the microbial growth state, may be influenced by thecurrent state of the microbes (ex. exponential growth, stationarygrowth, unstressed, stressed).

Inoculation of the production fermenter(s) can impact growth kineticsand cellular activity. The initial state of the bioreactor system mustbe optimized to facilitate successful and consistent production. Thefraction of seed culture to total medium (e.g. a percentage) has adramatic impact on growth kinetics. The range may be 1-5% of thefermenter's working volume. The initial pH of the culture medium may bedifferent from the process set-point. pH stress may be detrimental atlow cell concentration; the initial pH may be between pH 7.5 and theprocess set-point. Agitation and gas flow into the system duringinoculation may be different from the process set-points. Physical andchemical stresses due to both conditions may be detrimental at low cellconcentration.

Process conditions and control settings may influence the kinetics ofmicrobial growth and cellular activity. Shifts in process conditions maychange membrane composition, production of metabolites, growth rate,cellular stress, etc. Optimal temperature range for growth may vary withstrain. The range may be 20-40 ° C. Optimal pH for cell growth andperformance of downstream activity may vary with strain. The range maybe pH 5-8. Gasses dissolved in the medium may be used by cells formetabolism. Adjusting concentrations of 0₂, CO₂, and N₂ throughout theprocess may be required. Availability of nutrients may shift cellulargrowth. Microbes may have alternate kinetics when excess nutrients areavailable.

The state of microbes at the end of a fermentation and during harvestingmay impact cell survival and activity. Microbes may be preconditionedshortly before harvest to better prepare them for the physical andchemical stresses involved in separation and downstream processing. Achange in temperature (often reducing to 20-5° C.) may reduce cellularmetabolism, slowing growth (and/or death) and physiological change whenremoved from the fermenter. Effectiveness of centrifugal concentrationmay be influenced by culture pH. Raising pH by 1-2 points can improveeffectiveness of concentration but can also be detrimental to cells.Microbes may be stressed shortly before harvest by increasing theconcentration of salts and/or sugars in the medium. Cells stressed inthis way may better survive freezing and lyophilization duringdownstream.

Separation methods and technology may impact how efficiently microbesare separated from the culture medium. Solids may be removed usingcentrifugation techniques. Effectiveness of centrifugal concentrationcan be influenced by culture pH or by the use of flocculating agents.Raising pH by 1-2 points may improve effectiveness of concentration butcan also be detrimental to cells. Microbes may be stressed shortlybefore harvest by increasing the concentration of salts and/or sugars inthe medium. Cells stressed in this way may better survive freezing andlyophilization during downstream. Additionally, Microbes may also beseparated via filtration. Filtration is superior to centrifugationtechniques for purification if the cells require excessive g-minutes tosuccessfully centrifuge. Excipients can be added before afterseparation. Excipients can be added for cryo protection or forprotection during lyophilization. Excipients can include, but are notlimited to, sucrose, trehalose, or lactose, and these may bealternatively mixed with buffer and anti-oxidants. Prior tolyophilization, droplets of cell pellets mixed with excipients aresubmerged in liquid nitrogen.

Harvesting can be performed by continuous centrifugation. Product may beresuspended with various excipients to a desired final concentration.Excipients can be added for cryo protection or for protection duringlyophilization. Excipients can include, but are not limited to, sucrose,trehalose, or lactose, and these may be alternatively mixed with bufferand anti-oxidants. Prior to lyophilization, droplets of cell pelletsmixed with excipients are submerged in liquid nitrogen.

Lyophilization of material, including live bacteria, begins with primarydrying. During the primary drying phase, the ice is removed. Here, avacuum is generated and an appropriate amount of heat is supplied to thematerial for the ice to sublime. During the secondary drying phase,product bound water molecules are removed. Here, the temperature israised higher than in the primary drying phase to break anyphysico-chemical interactions that have formed between the watermolecules and the product material. The pressure may also be loweredfurther to enhance desorption during this stage. After the freeze-dryingprocess is complete, the chamber may be filled with an inert gas, suchas nitrogen. The product may be sealed within the freeze dryer under dryconditions, preventing exposure to atmospheric water and contaminants.

Example 25 A Mouse Melanoma Model

Female 6-8 week old C57B1/6 mice are obtained from Taconic (Germantown,NY). 100,000 B16-F10 (ATCC CRL-6475) tumor cells are resuspended insterile PBS containing 50% Matrigel and inoculated in a 100ul finalvolume into one hind flank (the first flank) of each mouse. Treatmentwith Veillonella Strains is initiated at some point following tumor cellinoculation at varied doses and at defined intervals. For example, somemice receive between 1-5×10{circumflex over ( )}9 CFU (100 μl finalvolume) per dose. Possible routes of administration include oral gavage(p.o.), intravenous injection, intratumoral injection (IT) orperitumoral or subtumoral or subcutaneous injection. In order to assessthe systemic anti-tumoral effects of Veillonella treatment, additionalmice may be inoculated with tumor cells in the contralateral (untreated,second) flank prior to IT, peritumoral, or subtumoral treatment withVeillonella in the first flank.

Some mice may receive Veillonella (p.o.) on day 1 (the day followingtumor cell injection). Other mice may receive seven (7) consecutivedoses of a bacterial strain (one dose per day on days 14-21). Other micereceive daily dosing or, alternatively, some mice receive dosing everyother day. Alternatively, mice are randomized into various treatmentgroups at a defined timepoint (e.g. on day 13) or when the tumors reacha certain size (e.g. 100 mm³) and treatment is then initiatedaccordingly. For example, when tumor volumes reach an average of 100mm³(approximately 10-12 days following tumor cell inoculation), animals aredistributed into groups and treated with either vehicle or a bacterialstrain (p.o. or IT). Some additional groups of mice may be treated withan additional cancer therapeutic or appropriate control antibody. Oneexample of a cancer therapeutic that may be administered is an inhibitorof an immune checkpoint, for example anti-PD-1, anti-PD-L1, or othertreatment that blocks the binding of an immune checkpoint to itsligand(s). Checkpoint inhibitors anti-PD-1 and anti-PD-L1 may beformulated in PBS and administered intraperitoneally (i.p.) in effectivedoses. For example, mice are given 100ug of anti-PD-1 (i.p.) every fourdays starting on day 1, and continuing for the duration of the study.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5g/L), ampicillin (1.0g/L), gentamicin(1.0g/L) and amphotericin B (0.2g/L) are added to the drinking water,and antibiotic treatment is halted at the time of treatment or a fewdays prior to treatment. Some mice are inoculated with tumor cellswithout receiving prior treatment with antibiotics.

At various timepoints, mice are sacrificed and tumors, lymph nodes, orother tissues may be removed for ex vivo flow cytometric analysis usingmethods known in the art. For example, tumors are dissociated using aMiltenyi tumor dissociation enzyme cocktail according to themanufacturer's instructions. Tumor weights are recorded and tumors arechopped then placed in 15 ml tubes containing the enzyme cocktail andplaced on ice. Samples are then placed on a gentle shaker at 37° C. for45 minutes and quenched with up to 15ml complete RPMI. Each cellsuspension is strained through a 70 μm filter into a 50 ml falcon tubeand centrifuged at 1000 rpm for 10 minutes. Cells are resuspended inFACS buffer and washed to remove remaining debris. If necessary, samplesare strained again through a second 70 μm filter into a new tube. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Rorγt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, WICK CD206, CD40, CSF1R, PD-L1, Gr-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-lb, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out immune cells obtainedfrom lymph nodes or other tissue, and/or on purifiedCD45+tumor-infiltrated immune cells obtained ex vivo. Finally,immunohistochemistry is carried out on tumor sections to measure Tcells, macrophages, dendritic cells, and checkpoint molecule proteinexpression.

Rather than being sacrificed, some mice may be rechallenged with tumorcell injection into the contralateral flank (or other area) to determinethe impact of the immune system's memory response on tumor growth.

In mice receiving the MCD (NASH-inducing) diet, orally administeredVeillonella was efficacious in reducing the NAS score compared tovehicle and no treatment groups (negative controls) (FIG. 16).Veillonella reduced the fibrosis score in treated mice (FIG. 17).Veillonella reduced hepatic total cholesterol (FIG. 18) and hepaticTriglycerides (FIG. 19).

Example 26 A Colorectal Carcinoma Model

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown,NY). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) wereresuspended in sterile PBS and inoculated in the presence of 50%Matrigel. CT-26 tumor cells were subcutaneously injected into one hindflank of each mouse. When tumor volumes reached an average of 100mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups: 1) Vehicle; 2) VeillonellaStrains 3) anti-PD-1 antibody. Antibodies were administeredintraperitoneally (i.p.) at 200 μg/mouse (100 μl final volume) everyfour days, starting on day 1, for a total of 3 times (Q4D×3) andVeillonella EVs (5 μg) were intravenously (i.v.) injected every thirdday, starting on day 1 for a total of 4 times (Q3D×4). Both Veillonellagroups showed tumor growth inhibition greater than that seen in theanti-PD-1 group (FIGS. 12 and 13).

Another example, when tumor volumes reached an average of 100 mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups: 1) Vehicle; 2) Burkholderiapseudomallei; and 3) anti-PD-1 antibody. Antibodies were administeredintraperitoneally (i.p.) at 200 μg/mouse (100 μl final volume) everyfour days, starting on day 1, and Burkholderia pseudomallei EVs (5 μg)were intravenously (i.v.) injected daily, starting on day 1 until theconclusion of the study. The Burkholderia pseudomallei group showedtumor growth inhibition greater than that seen in the anti-PD-1 group(FIGS. 20, and 21).

Another example, when tumor volumes reached an average of 100 mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups: 1) Vehicle; 2) NeisseriaMeningitidis EVs isolated from the Bexsero® vaccine; and 3) anti-PD-1antibody. Antibodies were administered intraperitoneally (i.p.) at200ug/mouse (100ul final volume) every four days, starting on day 1, andNeisseria Meningitidis bacteria (about 1.1×10²) were administeredintraperitoneally (i.p.) daily, starting on day 1 until the conclusionof the study. The Neisseria Meningitidis group showed tumor growthinhibition greater than that seen in the anti-PD-1 group (FIGS. 22, and23).

TABLE 4 Significance test comparing tumor volume in treatment groups vs.control groups on day 11. T-test (two-tailed, unpaired, welch-corrected)calculated in GraphPad. Comparison P value Summary IP Vehicle vs.Neisseria 0.0004 *** Meningitidis EVs.

Example 27 Efficacy of EVs Varies Based on Source Microbe, Dose, andRoute of Administration

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown,N.Y.). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) wereresuspended in sterile PBS and inoculated in the presence of 50%Matrigel. CT-26 tumor cells were subcutaneously injected into one hindflank of each mouse. When tumor volumes reached an average of 100mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups as highlighted in Table 5.

TABLE 5 Treatment Groups Group Treatment Dose/Route/Schedule 1 IVVehicle (PBS) N/A/IV/Q3Dx4 2 PO Vehicle (sucrose) N/A/PO/QD 3 Anti-PD-1200 μg/IP/Q4Dx3 4 Veillonella parvula EV 10 μg/IV/Q3Dx4 5 Veillonellaparvula EV 5 μg/IV/Q3Dx4 6 Veillonella parvula EV 2 μg/IV/Q3Dx4 7Veillonella tobetsuensis EV 75 μg/PO. QD 8 Veillonella tobetsuensis EV 5μg/IV/Q3Dx4

As noted in the table, antibodies were administered intraperitoneally(i.p.) at 200 μg/mouse (100 μl final volume) every four days, startingon day 1, for a total of 3 times (Q4D×3) and EVs when administeredintravenously (i.v.) were injected every third day, starting on day 1for a total of 4 times (Q3D×4). The treatment groups administered bymouth (p.o.) were administered daily (QD). Efficacy of Veillonella EVsvaries based on source microbe, dose, and route of administration (FIGS.14 and 15).

INCORPORATION BY REFERENCE

All publications patent applications mentioned herein are herebyincorporated by reference in their entirety as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. In case of conflict, thepresent application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A pharmaceutical composition comprising isolated bacterialextracellular vesicles (EVs).
 2. A pharmaceutical composition comprisingbacterial extracellular vesicles (EVs) and bacteria.
 3. Thepharmaceutical composition of claim 2, wherein at least, about, or nomore than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% of the total EV and bacteria particles in the pharmaceuticalcomposition are EVs.
 4. The pharmaceutical composition of claim 2wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% of the total EV and bacteriaparticles in the pharmaceutical composition are bacteria.
 5. Thepharmaceutical composition of claim 2 wherein at least, about, or nomore than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, Application No. Not Yet Assigned 5 Docket No.: ETB-01002Preliminary Amendment 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% of the total EV and bacteria protein in thepharmaceutical composition is EV protein.
 6. In some embodiments, atleast, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the total EV and bacteria protein in thepharmaceutical composition is a bacteria protein.
 7. The pharmaceuticalcomposition of claim 2 wherein, at least, about, or no more than 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total EVand bacteria lipids in the pharmaceutical composition are EV lipids. 8.The pharmaceutical composition of claim 2 wherein, at least, about, orno more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% of the total EV and bacteria lipids in the pharmaceuticalcomposition are bacteria lipids.
 9. A pharmaceutical compositioncomprising bacteria isolated from EVs.
 10. The pharmaceuticalcomposition of claim 2, wherein the composition comprises live, killed,or attenuated bacteria. 11-75. (canceled)
 76. A method of treating adisease in a subject comprising administering to the subject apharmaceutical composition according to claim
 1. 77. (canceled)
 78. Amethod of treating cancer in a subject comprising administering to thesubject a pharmaceutical composition according to claim
 1. 79. A methodof augmenting a microbiome in a subject who has cancer, the methodcomprising administering to the subject a pharmaceutical compositionaccording to an claim 1 to the subject such that the EVs and/or bacteriaare added to a niche in the subject.
 80. A method of depleting a tumorof cancer-associated bacteria in a subject, the method comprisingadministering to the subject a pharmaceutical composition according toclaim 1 to the subject such that the EVs and/or bacteria are added to aniche in the subject.
 81. A method of changing a tumor microbiome in asubject, the method comprising administering to the subject apharmaceutical composition according to claim 1 to the subject such thatthe EVs and/or bacteria are added to a niche in the subject.
 82. Amethod of changing a mesenteric lymph node microbiome in a subject, themethod comprising administering to the subject a pharmaceuticalcomposition according to claim 1 to the subject such that the EVs and/orbacteria are added to a niche in the subject.
 83. A method of changingan antigen presentation by dendritic cells in a subject, the methodcomprising administering to the subject a pharmaceutical compositionaccording to claim 1 to the subject such that the EVs and/or bacteriaare added to a niche in the subject.
 84. A method of activatingepithelial cells in a subject, the method comprising administering tothe subject a pharmaceutical composition according to claim 1 to thesubject such that the EVs and/or is added to a niche in the subject.85-105. (canceled)
 106. A method of treating an immune disorder in asubject comprising administering to the subject a pharmaceuticalcomposition according to claim
 1. 107-115. (canceled)
 116. A method ofgenerating an engineered bacterium comprising introducing into thebacterium a modification that results in the increased production ofEVs. 117-126. (canceled)
 127. A method of generating an engineeredbacterium comprising introducing into the bacterium a modification thatresults the production of EVs with an improved therapeutic property bythe bacterium. 128-146. (canceled)
 147. A modified bacterium generatedaccording to the method of claim
 127. 148. A method of culturing abacterium for improved EV production, the method comprising growing thebacteria under stress-inducing growth conditions. 149-160. (canceled)161. A bioreactor comprising a modified bacterium according to claim147.